Novel immunostimulating vector system

ABSTRACT

A method of treating cancer, a viral infection and/or an immune system disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a vector, wherein the vector comprises (a) nucleic acid sequences encoding 4-1BB ligand (4-1BBL), single chain IL-12 (scIL-12) and IL-2, and (b) at least one regulatory nucleic acid sequence providing for an increased expression level of 4-1BBL as compared to the expression levels of scIL-12 and IL-2, and other related methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of pending U.S.patent application Ser. No. 16/079,331, filed Aug. 23, 2018, which isthe U.S. national phase of International patent Application No.PCT/EP2017/054216, filed on Feb. 23, 2017, which claims the benefit ofEuropean Application No. 16157423.1, filed Feb. 25, 2016, thedisclosures of which are incorporated herein by reference in theirentireties.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: 38,217 bytes ASCII (Text) file named “752005_ST25.bd,”created Apr. 1, 2021.

TECHNICAL FIELD

The present disclosure relates generally to the field of immunology, andmore specifically to the field of immunotherapy. In particular, thepresent disclosure relates to a novel vector system forimmunostimulation and methods of using same in immunotherapy. The novelvector system is characterized by one or more vectors comprising nucleicacid sequences encoding 4-1BB ligand (4-1BBL, CD137 ligand), singlechain IL-12 (scIL-12) and IL-2.

BACKGROUND

The immune system provides the means for recognizing and destroyingforeign invaders, such as bacteria or viruses, as well as damaged,diseased, or abnormal cells in the body, including cancer cells. Primaryplayers in immune system responses include macrophages and naturalkiller cells, which provide a general, or nonspecific, level of immuneprotection. Other cell types, including cytotoxic T lymphocytes (CTLs)and B lymphocytes, act against specific targets. Immune responsesinclude humoral responses, in which B cells produce antigen-specificantibodies, and cell-mediated responses, in which antigens orantigen-bearing cells are recognized and destroyed by various types of Tcells by using a variety of different mechanisms. Cell-mediated immuneresponses including a CTL response are considered to be key to theelimination of tumor cells and virus-infected cells.

It is generally believed that the natural capacity of the immune systemto detect and destroy abnormal cells prevents the development of manycancers. Nevertheless, some cancer cells have developed strategies toevade destruction by the immune system. For example, several differentmechanisms exist which cancer cells can use to suppress immuneresponses. They can also undergo genetic changes that lead to the lossof cancer-associated antigens, making them less “visible” to the immunesystem. Similar considerations apply with respect to different viruses,which have also adopted immune evasion strategies, leading to a failureof the host's immune system to control viral infection.

The goal of immunotherapy is to overcome these barriers to an effectiveimmune response. Immunotherapy-based biological therapies restore orincrease the activities of specific immune-system components orcounteract immunosuppressive signals produced by cancer cells or duringviral infectious diseases. Tumor cells, among others, are killed by CTLsin an antigen-specific manner. Thus, agents that promote T-cellactivation and impart strong cytolytic and inflammatory properties areideal candidates for enhancing tumor-specific immunity. New forms oftherapy continue to be needed, and one major avenue ofimmunotherapeutics is based on gene transfer technology. Gene therapyhas been established as a way to deliver immune therapy. Non-replicatingviruses and viral vectors were originally proposed 20 years ago asanticancer agents using, inter alia, immune activating (e.g., IL-2)modalities (see, for example, Crofts and Krimsky, 2005, Hum Gene Ther.16: 169-177). WO 2004/035799 describes an adenoviral vector comprisingnucleic acid sequences, which code for single chain IL-12 (scIL-12), thecostimulatory protein 4-1BB ligand (4-1BBL), and IL-2, for gene therapyin the treatment of infectious diseases and cancer.

The present disclosure relates to a novel vector-based immunotherapy,which represents an improvement over prior gene therapy approacheseffective for converting an inactive into an active immunemicroenvironment and thereby treating cancer and viral infections.

SUMMARY OF THE DISCLOSURE

The present disclosure provides a vector comprising nucleic acidsequences of genes encoding 4-1BB ligand (4-1BBL), single chain IL-12(scIL-12) and IL-2, wherein the vector provides for an increasedexpression of 4-1BBL as compared to the expression levels of scIL-12 andIL-2. Specifically, the nucleic acid sequences encoding 4-1BBL, scIL-12and IL-2 are organized in the vector in 5′ to 3′ orientation in asequential order 1, 2, 3, with the proviso that the gene encodingscIL-12 is not at position 1. The present disclosure also providesmethods and uses of the novel vector for converting an inactive into anactive immune microenvironment and thereby treating cancer or infectiousdiseases. Methods and compositions of the present disclosure include theconstruction and verification of the claimed viral vector that elicitsan immune response against cancer cells and viral infections to enhanceand/or stimulate immunity against cancer and viral infections.

Aspects of the disclosure include:1. Vector comprising nucleic acid sequences encoding 4-1BB ligand(4-1BBL), single chain IL-12 (scIL-12) and IL-2, and further comprisingat least one regulatory nucleic acid sequence, preferably a promotersequence, providing for an increased expression level of 4-1BBL ascompared to the expression levels of scIL-12 and IL-2.2. The vector of item 1, wherein the expression level of 4-1BBL isincreased as compared to the expression level of 4-1BBL obtained by theexpression construct of vector Im01 (FIG. 20).3. The vector of item 1 or 2, wherein the expression level of scIL-12 isdecreased and/or the expression level of IL-2 is increased as comparedto the expression levels of scIL-12 and/or IL-2 obtained by theexpression construct of vector Im01 (FIG. 20).4. The vector of any one of items 1 to 3, wherein the nucleic acidsequences encoding 4-1BBL, scIL-12 and IL-2 are organized in 5′ to 3′orientation in a sequential order 1, 2, 3, with the proviso that thenucleic acid sequence encoding scIL-12 is not at position 1.5. The vector of any one of items 1 to 4, wherein the vector is any oneof an adenoviral vector, an adeno-associated virus vector, a lentiviralvector, a herpes simplex virus vector, a pox virus vector, an RNAvector, a plasmid vector, a nanoparticle vector, and naked DNA.6. The vector of any one of items 1 to 5, wherein the nucleic acidsequence encoding 4-1BBL is human cDNA, the nucleic acid sequenceencoding scIL-12 is human cDNA, and/or the nucleic acid sequenceencoding IL-2 is human cDNA.7. The vector of any one of items 1-6, wherein the nucleic acid sequenceencoding 4-1BBL shows at least 70% sequence identity to the nucleic acidsequence of SEQ ID NO: 1, wherein the variant nucleic acid sequenceencodes a 4-1BBL protein capable of specifically binding activated Tcells.8. The vector of any one of items 1-6, wherein the nucleic acid sequenceencoding IL-2 shows at least 70% sequence identity to the nucleic acidsequence of SEQ ID NO: 3, wherein the variant nucleic acid sequenceencodes an IL-2 protein having immune stimulating activity.9. The vector of any one of items 1-6, wherein the nucleic acid sequenceencoding scIL-12 shows at least 70% sequence identity to the nucleicacid sequence of SEQ ID NO: 5, wherein the variant nucleic acid sequenceencodes a scIL-12 protein having immune stimulating activity.10. The vector of any one of items 1-9, wherein the nucleic acidsequences encoding scIL-12 and IL-2 are located downstream of thenucleic acid sequence encoding 4-1BBL.11. The vector of item 10, wherein the nucleic acid sequence encodingIL-2 is located downstream of the nucleic acid sequence encoding 4-1BBL,and the nucleic acid sequence encoding scIL-12 is located downstream ofthe nucleic acid sequence encoding IL-2.

12. The vector of item 10 or 11, wherein a promoter is located upstreamof the nucleic acid sequence encoding 4-1BBL, but not upstream of thenucleic acid sequences encoding scIL-12 and/or IL-2.

13. The vector of any one of items 1-12, wherein the nucleic acidsequences encoding 4-1BBL, scIL-12 and IL-2 are linked by internalribosomal entry sites (I RES).14. A cancer cell or an immune cell, transduced or transfected with thevector of any one of items 1-13.15. A medicament comprising the vector of any one of items 1-13 or thecancer or immune cell of item 14.16. The vector of any one of items 1-13, the cancer or immune cell ofitem 14, or the medicament of item 15, for use in, or for use in amethod of, treating cancer, a viral infection and/or an immune systemdisorder.17. The vector for use according to item 16, or the cancer or immunecell for use according to item 16, or the medicament for use accordingto item 16, wherein the cancer is any one of bladder cancer, breastcancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, coloncancer, melanoma, malignant melanoma, ovarian cancer, brain cancer,primary brain carcinoma, head-neck cancer, glioma, glioblastoma, livercancer, non-small cell lung cancer, head or neck carcinoma, breastcarcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma,Wilms' tumor, cervical carcinoma, testicular carcinoma, bladdercarcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma,prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma,esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma,renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma,malignant pancreatic insulinoma, malignant carcinoid carcinoma,choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervicalhyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocyticleukemia, acute myelogenous leukemia, chronic myelogenous leukemia,chronic granulocytic leukemia, acute granulocytic leukemia, hairy cellleukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, mesothelioma, osteogenicsarcoma, primary macro globulinemia, and retinoblastoma.18. The vector of any one of items 1-13, or the cancer or immune cell ofitem 14, or the medicament of item 15, for use in, or for use in amethod of, preventing or treating cancer metastasis.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of transgene expression of IL-12, IL-2 and4-1BBL and IFN-γ response of murine and human Im01. MOI (multiplicity ofinfection) numbers indicated in [brackets]; “m”=murine; “hu”=human.

FIG. 2 shows a schematic gene map of the shuttle plasmid pE1.1 Im02. ThecDNAs of the three therapeutic genes of human 4-1BBL (CD137L), IL-2 andsingle-chain IL-12 are organized in a tricistronic construct linked byinternal ribosomal entry sites (IRES) and driven by a Cytomegalovirus(CMV) promoter. Transcript polyadenylation is induced by a SV40 derivedsignal. The expression cassette for Im02 is illustrated as a precursortransfer plasmid based on plasmid pE1.1, suitable for sub-cloning into aplasmid carrying, e.g., adenoviral vector DNA.

FIG. 3 shows a comparison of transgene expression and IFN-γ response ofIm02 and the earlier vector Im01. MOI (multiplicity of infection)numbers indicated in [brackets]. Indicated data points are the mean offour individual donors each with four replicates.

FIG. 4 shows a comparison of Im02 and Im01 in a bladder tissue-basedmodel. “T”=bladder tumor tissue; “B”=normal bladder tissue.

FIG. 5 shows a comparison of Im02 and Im01 at different dose levels.Dosis ivp means dosis of infectious virus particles. “T”=bladder tumortissue; “B”=normal bladder tissue.

FIG. 6 shows a heat-plot illustrating the activation of immune celltypes in a co-culture of human bladder RT-4 carcinoma cells with humanPBMCs. Subsequent rows show basic and activated (“act”) immune celltypes. RT-4 cells without vector (“control”) and cells transduced withan empty adenovirus vector (“Ad0”) were used as controls. Th=T helpercells; Tc=cytotoxic T cells (CD8+ T cells); PC=plasma cells; NK=naturalkiller cells; mono=monocytes; DC=dendritic cells; neutro=neutrophils.

FIG. 7 shows histologic examination after Hematoxylin and Eosin stainingof samples stimulated with Im02 or Ad0 or without treatment as control.A: Bladder tumor tissue without culture; B: Bladder tumor tissue, 10⁸ivp Ad0, 6 days after treatment; C: Bladder tumor tissue, 10⁸ ivp Im02,6 days after treatment.

FIG. 8 shows histological examination after Hematoxylin and Eosinstaining of samples stimulated with or without Im02. Upper panel:Urothelial tumor tissue without culture, lower panel: Bladder tumortissue, stimulated with 10⁸ ivp Im02, 6 days after treatment.

FIG. 9 shows target cell analysis and uptake mechanism by transmissionelectron microscopy. Panel 1: Overview (right image), and Detail (A):particle uptake without vesicle by an unknown cell type. Detail (B),white: vesicular particle uptake by a cell of Langerhanscell/macrophage/dendritic cell morphology. Panel 2: Overview (B) andDetail (A): vesicular adenoviral particle uptake by a cell of Langerhanscell/macrophage/dendritic cell morphology. Panel 3: Overview (B) andDetail (A): Adenoviral particle uptake by a strong vesicular structureby a cell of lymphocyte morphology. Panel 4: Overview (B) and Detail(A): Adenoviral particle uptake without vesicle by a cell of fibroblastmorphology. Panel 5: Overview (B) and Detail (A): Large groups ofadenoviral particles without vesicle taken up by an unknown target celltype. Panel 6: Overview: Adenoviral particles without vesicle coating(white arrows) between abundant vesicular structures in a target cellwith morphological features of an epithelial or tumor cell. Detail (A):Early endocytosis figure of an adenoviral particle attached to theplasma membrane. Detail (B): Pinocytosis of an adenoviral particle in alarge vesicle containing also other non-viral structures.

FIG. 10 shows a comparison of differential expression in normal bladderand bladder tumor samples. Im02 means the adenoviral vector as describedin Example 2. Ad0 means empty adenoviral vector, used as control.n=number of bladder/tumor samples.

FIG. 11 shows the examination of different transfectants in cellculture. GFP=green fluorescent protein.CAR=Coxsackie-Adenovirus-Receptor. MOI=multiplicity of infection(infectious viral particles per target cell).

FIG. 12 shows effects of protamine sulfate on transgene expression andIFN-γ induction. Im02 means the adenoviral vector as described inExample 2; Ad0=empty adenoviral vector, used as a control.ivp=infectious viral particles. “T”=bladder tumor tissue; “B”=normalbladder tissue.

FIG. 13 shows the nucleotide and amino acid sequence of human 4-1BBL(CD137L).

FIG. 14 shows the nucleotide and amino acid sequence of human IL-2.

FIG. 15 shows the nucleotide and amino acid sequence of humansingle-chain IL-12 (scIL-12) comprising the p40 and p35 subunits ofhuman IL-12 linked by a linker. The linker sequence is shown in boldfaceand underlined in SEQ ID NO: 5 and 6, respectively.

FIG. 16 shows the nucleotide and amino acid sequences of the 35 kDa and40 kDa subunits of human IL-12.

FIG. 17 shows the nucleotide sequence (SEQ ID NO: 11) of the shuttlevector hu pE1.1 Im02 depicted in FIG. 2. The nucleotide sequence has atotal of 7,845 bp. The CMV promoter, human 4-1BBL, EMCV IRES, humanIL-2, PV IRES, human scIL-12, and SV40polyA can be identified in thenucleotide sequence of SEQ ID NO: 11 as follows: CMV promoter: bp484-1,059; human 4-1BBL: bp 1,080-1,844; EMCV IRES: bp 1,885-2,388;human IL-2: bp 2,409-2,870; PV IRES: bp 2,914-3,545; human scIL-12: p40subunit bp 3,581-4,564, linker bp 4,565-4,609, p35 subunit bp4,610-5,203; and SV40polyA: bp 5,271-5,510.

FIG. 18 shows a schematic overview and the nucleotide sequence of theexpression cassette comprising CMV, human 4-1BBL, EMCV IRES, human IL-2,PV IRES, human scIL-12, and SV40polyA as contained in the shuttleplasmid hu pE1.1 Im02 depicted in FIG. 2 and in SEQ ID NO: 11 (FIG. 17),respectively.

FIG. 19 shows transgene expression of 4-1BBL, IL-2, and scIL-12 of Im02and of single-gene expressing vectors at different MOI, and IFN-γresponse. The axis at the bottom designated “% of 4-1BBL+ cells” meanscells that are positive for expression of 4-1BBL. The bar on theleft-hand side indicates the combination of IL-12 and IL-2, each at [5]MOI, with different MOIs of 4-1BBL ([10], [25], [50], and [100]). Im02means the adenoviral vector as described in Example 2. MOI=multiplicityof infection (i.e., infectious virus particles per target cell).Transgene expression of 4-1BBL, IL-2 and scIL-12, and IFN-γ response ofIm02, and combinations of single-gene expressing vectors reveal thatIFN-γ expression is dependent on increasing 4-1BBL levels.

FIG. 20 shows a schematic gene map of the shuttle plasmid pE1.1 Im01.The cDNAs of the three therapeutic genes of human 4-1BBL (CD137L), IL-2and single-chain IL-12 are organized in a tricistronic construct linkedby internal ribosomal entry sites (IRES) and driven by a Cytomegalovirus(CMV) promoter. Transcript polyadenylation is induced by a SV40 derivedsignal. The expression cassette for Im01 is illustrated as a precursortransfer plasmid based on plasmid pE1.1, suitable for sub-cloning into aplasmid carrying, e.g., adenoviral vector DNA.

DETAILED DESCRIPTION OF THE DISCLOSURE

As used herein and in the appended claims, the singular forms “a”,“and”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an antigen”includes a plurality of such antigens, and reference to “a cell” or “thecell” includes reference to one or more cells and equivalents thereof(e.g., plurality of cells) known to those skilled in the art, and soforth. Similarly, reference to “a compound” or “a composition” includesa plurality of such compounds or compositions, and refers to one or morecompounds or compositions, respectively, unless the context clearlydictates otherwise. The term “about” when referring to a number or anumerical range means that the number or numerical range referred to isan approximation within experimental variability (or within statisticalexperimental error), and thus the number or numerical range may varybetween 1% and 15% of the stated number or numerical range. The term“comprising” (and related terms such as “comprise” or “comprises” or“having” or “including”) is not intended to exclude that in othercertain embodiments, for example, an embodiment of any composition ofmatter, composition, method, or process, or the like, described herein,may “consist of” or “consist essentially of” the described features.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The term “codon optimized sequences” generally refers to nucleotidesequences that have been optimized for a particular host species byreplacing any codons having a usage frequency of less than about 20%.Nucleotide sequences that have been optimized for expression in a givenhost species by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as “expression enhancedsequences”.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. The regulatory sequence may behomologous or heterologous to the desired gene sequence. For example, awide range of promoters may be utilized, including viral or mammalianpromoter as described herein. The promoter is oriented relative to a DNAsequence such that it is capable of initiating transcription of the saidDNA sequence. The term “regulatory nucleic acid sequence” referscollectively to promoter sequences, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, enhancers and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell. One skilled in the art can readily identify regulatory nucleicacid sequence from public databases and materials. Furthermore, oneskilled in the art can identify a regulatory sequence that is applicablefor the intended use, for example, in vivo, ex vivo, or in vitro.Preferably, the term “regulatory nucleic acid sequence” refers to apromoter or promoter sequence.

In various embodiments, the term “regulatory nucleic acid sequence” alsorefers to IRES sequences (internal ribosomal entry sites). This appliesin particular to various preferred embodiments, in which the term“regulatory nucleic acid sequences” encompasses one promoter pertricistronic expression cassette/vector containing the nucleic acidsequences encoding 4-1BBL, scIL-12 and IL-2, and further encompasses anIRES sequence for each cistron that is not localized immediatelydownstream of the promoter. The combination of a promoter and IRESsequences is considered, and has been demonstrated, to provide for animproved expression level or expression rate of 4-1BBL as compared tothe expression levels or expression rates of IL-2 and scIL-12 in atricistronic expression cassette/vector containing the nucleic acidsequences encoding 4-1BBL, IL-2 and scIL-12 (in this order) with apromoter for 4-1BBL, and IRES sequences for IL-2 and scIL-12.

The phrases “operatively positioned,” “operatively linked,” “undercontrol” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence. A promoter may or may not be used inconjunction with an “enhancer”, which refers to a cis-acting regulatorysequence involved in the transcriptional activation of a nucleic acidsequence.

WO 2004/035799 describes an adenoviral vector comprising an expressionconstruct comprising the genes for mouse/human single-chain IL-12(scIL-12), 4-1BB ligand (4-1BBL), and IL-2 in this order in 5′ to 3′orientation, i.e., the gene encoding scIL-12 at position 1, the geneencoding 4-1BBL at position 2, and the gene encoding IL-2 at position 3.This expression construct is shown in FIG. 1 of WO 2004/035799, and thecorresponding vector was named “Ad-3”. In the present disclosure, theinternal vector code for the said earlier vector “Ad-3” is “Im01”, seealso Example 1 of the present disclosure.

Transgene expression of scIL-12, 4-1BBL, and IL-2 of murine and humanIm01 has revealed that the expression level of the transgenes clearlyvaries between murine and human species, as shown in Example 1 and FIG.1 of the present disclosure. Notably, expression of Im01 carrying thegenes of human scIL-12, human 4-1BBL, and human IL-2 (“human Im01” or“hu Im01”) resulted in a marked increase in IL-12 as compared toexpression of Im01 carrying the genes of mouse scIL-12, mouse 4-1BBL,and mouse IL-2 (“mouse Im01” or “m Im01”). Thus, despite the same vectorarchitecture of mouse and human Im01 as regards the organization of theorder of the genes of scIL-12, 4-1BBL, and IL-2, marked differences inthe expression level of the transgenes were observed. At least some ofthese differences in transgene expression between mouse and human Im01were completely unexpected.

The vector provided by the present disclosure comprises a newarchitecture as regards the three genes encoding scIL-12, 4-1BBL, andIL-2. The novel vector provides for an increased expression of 4-1BBL ascompared to the expression levels of scIL-12 and IL-2. In particular,the vector provided by the present disclosure comprises an expressioncassette, wherein the three nucleic acid sequences (or the three genes)encoding scIL-12, 4-1BBL, and IL-2 are organized in 5′ to 3′ orientationin a sequential order 1, 2, and 3, with the proviso that the geneencoding scIL-12 is not at position 1. This novel vector architectureprovides for, inter alia, an increased expression of 4-1BBL as comparedto the arrangement of the same genes in human Im01, concurrent with adecrease of IL-12. Surprisingly, it has been found that this novelvector architecture leads to an increase in IFN-γ response (see Example3 and FIG. 3 and Example 11 and FIG. 19 of the present disclosure). Thesurprising effects provided for by the novel vector of the presentdisclosure have been proven to be particularly beneficial forimmunotherapy of patients in need of such therapy. Specifically, it hasbeen shown that the vector of the present disclosure is particularlyeffective for converting an inactive into an active immunemicroenvironment, thereby treating cancer or viral infections.Therefore, the vector of the present disclosure is particularly suitablefor use in cancer immunotherapy. For example, FIGS. 4 and 5 of thepresent disclosure demonstrate that the vector of the present disclosureexhibits an improved effect in immunostimulation in the tumormicroenvironment as compared to the earlier vector Im01, as explained incorresponding Examples 4 and 5. Furthermore, a transcriptome analysishas shown that the therapeutic gene expression profile of the vector ofthe present disclosure is unique and superior, in particular superiorover that of the earlier vector Im01 (see Example 6 and Tables 1 and 2).In addition, as shown in FIG. 6, gene expression analysis of theactivation of major immune cell types has shown that the activation of Thelper cells and cytotoxic T cells (CD8+ T cells) by the vector of thepresent disclosure is superior over the activation of the same immunecells by the earlier vector Im01 (see Example 7).

The novel vector of the present disclosure comprises three genesencoding 4-1BB ligand (4-1BBL), single-chain IL-12 (sc-IL12), and IL-2,wherein the said genes are organized in 5′ to 3′ orientation such thatthe gene encoding scIL-12 is not at the most upstream position of thesaid three genes. Thus, the novel vector of the present disclosurecomprises three genes encoding 4-1BB ligand (4-1BBL), single-chain IL-12(sc-IL12), and IL-2, wherein the said genes are organized in 5′ to 3′orientation in a sequential order 1, 2, and 3, with the proviso that thegene encoding scIL-12 is not at position 1. More specifically, thevector comprises nucleic acid sequences of genes encoding 4-1BBL,sc-IL12, and IL-2, wherein the said genes are organized in 5′ to 3′orientation in a sequential order 1, 2, and 3, with the proviso that thegene encoding scIL-12 is not at position 1. More specifically, thevector comprises nucleic acid sequences of genes encoding 4-1BBL,sc-IL12, and IL-2, wherein the said nucleic acid sequences of the saidgenes are organized in 5′ to 3′ orientation in a sequential order 1, 2,and 3, with the proviso that the nucleic acid sequence of the geneencoding scIL-12 is not at position 1.

In various embodiments of the present disclosure, the novel vectorcomprises an expression construct (or expression cassette) comprisingthree genes (more specifically nucleic acid sequences of three genes)encoding 4-1BB ligand (4-1BBL), single-chain IL-12 (sc-IL12), and IL-2,wherein the said genes are organized in the expression cassette suchthat the gene encoding scIL-12 is not at the most upstream position ofthe expression cassette. The most upstream position of the expressioncassette may be more specifically described as the position at the 5′end of the expression cassette. Also, the most upstream position of theexpression cassette may be more specifically described as the positionimmediately downstream of a promoter regulating transcription of thethree genes of the expression cassette. The promoter may be a promoterthat is part of the expression cassette, or a promoter upstream of theexpression cassette. The organization of the three transgenes the threegenes encoding 4-1BBL, sc-IL12, and IL-2) in the expression cassette maybe more specifically described to the effect that the nucleic acidsequences of the said three genes are organized in 5′ to 3′ orientationin a sequential order 1, 2, and 3, with the proviso that the nucleicacid sequence of the gene encoding scIL-12 is not at position 1 of thethree genes of the expression cassette.

In various embodiments of the present disclosure, the novel vector ofthe present disclosure comprises an expression construct (or expressioncassette) comprising three genes encoding 4-1BB ligand (4-1BBL),single-chain IL-12 (sc-IL12), and IL-2, wherein the said genes areorganized in 5′ to 3′ orientation in a sequential order 1, 2, and 3,with the proviso that the gene encoding scIL-12 is not at position 1.More specifically, the vector comprises an expression construct (orexpression cassette) comprising nucleic acid sequences of genes encoding4-1BBL, sc-IL12, and IL-2, wherein the said genes are organized in 5′ to3′ orientation in a sequential order 1, 2, and 3, with the proviso thatthe gene encoding scIL-12 is not at position 1. More specifically, thevector comprises an expression construct (or expression cassette)comprising nucleic acid sequences of genes encoding 4-1BBL, sc-IL12, andIL-2, wherein the said nucleic acid sequences of the said genes areorganized in 5′ to 3′ orientation in a sequential order 1, 2, and 3,with the proviso that the nucleic acid sequence of the gene encodingscIL-12 is not at position 1. In various embodiments, the expressioncassette or expression construct of the present disclosure comprises apromoter. The promoter may be more specifically described as promoterregulating transcription of the expression cassette (or expressionconstruct). In various embodiments, the expression cassette comprisesone promoter that is located upstream of (or at the 5′ end of) theexpression cassette (or upstream of the 5′ end of the expressioncassette).

As described herein, the “position 1” of the proviso clause mentionedabove may be more specifically described as position 1 of the saidsequential order 1, 2, and 3.

In various embodiments, organization of the three genes encodingscIL-12, 4-1BBL, and IL-2 in 5′ to 3′ orientation means organization ofthe three genes encoding scIL-12, 4-1BBL, and IL-2 in 5′ to 3′orientation relative to the expression cassette or expression constructof the present disclosure comprising the three genes encoding 4-1BBligand (4-1BBL), single-chain IL-12 (sc-IL12), and IL-2. Thus, 5′ to 3′orientation refers to the 5′ to 3′ orientation of the expressioncassette (or expression construct) or the promoter of the expressioncassette.

In various other embodiments of the present disclosure, organization ofthe three genes encoding scIL-12, 4-1BBL, and IL-2 in 5′ to 3′orientation means organization of the three genes encoding scIL-12,4-1BBL, and IL-2 in 5′ to 3′ orientation relative to a promoter, whichis not part of the expression cassette (or expression construct) of thedisclosure, but which is located upstream of the expression cassette (orexpression construct), i.e., upstream of the 5′ end of the expressioncassette (or expression construct). Here, 5′ to 3′ orientation refers tothe 5′ to 3′ orientation of the promoter upstream of the 5′ end of theexpression cassette (or expression construct). The promoter locatedupstream of the expression cassette (or upstream of the 5′ end of theexpression cassette) may be more specifically described as the promoterregulating transcription of the expression cassette (or expressionconstruct).

Also, 5′ to 3′ orientation may refer to the 5′ to 3′ orientation of thepromoter upstream of that gene of the three transgenes of the presentdisclosure (i.e., encoding scIL-12, 4-1BBL, and IL-2), which is at themost upstream position of the said three transgenes.

The vector provided by the present disclosure comprises a newarchitecture as regards the three genes encoding scIL-12, 4-1BBL, andIL-2 which can also be described to the effect that the said three genesare organized in 5′ to 3′ orientation in a sequential order 1, 2, and 3,with the proviso that the gene encoding scIL-12 is not at position 1,wherein said position 1 is downstream of a promoter, in particulardownstream of a promoter for expression of the said three genes. Thus,with respect to the expression cassette (or expression construct) of thepresent disclosure comprising the three genes encoding 4-1BB ligand(4-1BBL), single-chain IL-12 (sc-IL12), and IL-2, the said three genesare organized in 5′ to 3′ orientation in a sequential order 1, 2, and 3,with the proviso that the gene encoding scIL-12 is not at position 1,wherein said position 1 is downstream of a promoter, in particulardownstream of a promoter for transcription/expression of the saidexpression cassette (or expression construct).

As described herein, “located upstream” may be more specificallydescribed as “located directly upstream”. Furthermore, “located at the5′ end” or “located upstream of the 5′ end” may be more specificallydescribed as “located at the 5′ end of the transcription initiationsite” or “located upstream of the 5′ end of the transcription initiationsite”.

The novel vector of the present disclosure is capable of expressing thegenes (or nucleic acid sequences of the genes) encoding 4-1BBL, sc-IL12,and IL-2. Thus, more specifically, the novel vector of the presentdisclosure is an expression vector, even more specifically a recombinantexpression vector.

As described herein, in various embodiments, the term “nucleic acidsequences of genes encoding 4-1BBL, sc-IL12, and IL-2” means “nucleicacid sequences encoding 4-1BBL, sc-IL12, and IL-2”, and vice versa.Accordingly, in various embodiments, the term “nucleic acid sequence ofa gene encoding 4-1BBL” means “nucleic acid sequence encoding 4-1BBL”,and vice versa. Thus, in various embodiments, the term “nucleic acidsequence of a gene encoding scIL-12” means “nucleic acid sequenceencoding scIL-12”, and vice versa, and “nucleic acid sequence of a geneencoding IL-2” means “nucleic acid sequence encoding IL-2”, and viceversa. Likewise, in various embodiments, the term “nucleic acidsequences of genes encoding sc-IL12 and IL-2” means “nucleic acidsequences encoding sc-IL12 and IL-2”, and vice versa. As furtherdescribed herein, in various embodiments, the term “genes encoding4-1BBL, sc-IL12, and IL-2” means “nucleic acid sequences encoding4-1BBL, sc-IL12, and IL-2”, and vice versa. Accordingly, in variousembodiments, the term “gene encoding 4-1BBL” means “nucleic acidsequence encoding 4-1BBL”, and vice versa. Likewise, in variousembodiments, the term “genes encoding sc-IL12 and IL-2” means “nucleicacid sequences encoding sc-IL12 and IL-2”, and vice versa.

The novel vector of the present disclosure provides for a higherexpression of 4-1BBL as compared to the expression of scIL-12 and IL-2.Therefore, also provided by the present disclosure is a vector systemcomprising one or more vectors comprising nucleic acid sequencesencoding at least 4-1BB ligand (4-1BBL), single chain IL-12 (scIL-12)and IL-2, wherein the vector system provides for a higher expression of4-1BBL as compared to the expression of scIL-12 and IL-2. The higherexpression of 4-1BBL is regulated by different promoter strength of thepromoters regulating transcription of the three transgenes. The said oneor more vectors are expression vectors capable of expressing the nucleicacid sequences encoding 4-1BBL, scIL-12 and IL-2. More specifically, theexpression vectors are recombinant expression vectors. In variousembodiments, the vector system comprising one or more vectors providesfor the production of higher or increased levels of 4-1BBL at constantlevels of IL-12 and IL-2. In various embodiments, the vector systemcomprising one or more vectors can be used in a medical setting in orderto achieve that at least 5% of cancer cells or immune cells, transducedor transfected with the novel vector/vector system or a virus particledisclosed herein, are expressing 4-1BBL.

In one aspect, the present disclosure provides a vector systemcomprising one or more vectors comprising nucleic acid sequencesencoding at least 4-1BBL, scIL-12 and IL-2, wherein the vector systemprovides for the production of higher or increased levels of 4-1BBL atconstant levels of IL-12 and IL-2. This reflects the finding shown inFIG. 19 that IFN-γ expression is dependent on increasing 4-1BBL levels.Combinations of constant levels of IL-12 and IL-2, both at MOI [5], withincreasing levels of 4-1BBL up to MOI [100], lead to increasing IFN-γinduction at moderate IL-12 levels, as shown in FIG. 19. In variousembodiments, the vector system of the present disclosure may be used ina medical setting, preferably treatment of cancer, with increased levelsof 4-1BBL at constant levels of IL-12 and IL-2. Specifically, the vectorsystem may be used in a medical setting with a vector encoding 4-1BBL atMOI [10] and a vector encoding IL-2 and IL-12 at MOI [5]. Also, thevector system may be used in a medical setting with a vector encoding4-1BBL at MOI [10] and two vectors each encoding IL-2 and IL-12,respectively, at MOI [5]. In various embodiments, the vector system maybe used in a medical setting with a vector encoding 4-1BBL at MOI [25]and one or two vectors encoding IL-2 and IL-12 at MOI [5]. In variousother embodiments, the vector system may be used in a medical settingwith a vector encoding 4-1BBL at MOI [50] and one or two vectorsencoding IL-2 and IL-12 at MOI [5]. In various further embodiments, thevector system may be used in a medical setting with a vector encoding4-1BBL at MOI [100] and one or two vectors encoding IL-2 and IL-12 atMOI [5]. Preferably, the MOI applied for the vector encoding 4-1BBLachieves that at least 5% of cancer cells or immune cells, transduced ortransfected with the novel vector/vector system or a virus particledisclosed herein, are expressing 4-1BBL.

The medical setting referred to above encompasses, without being limitedthereto, the treatment of cancer or viral infections. Preferably, themedical setting means the treatment of cancer, more preferably thetreatment of solid cancers or solid tumors.

The novel vector system of the present disclosure is capable ofexpressing the genes (or nucleic acid sequences of the genes) encoding4-1BBL, sc-IL12, and IL-2. Thus, more specifically, the novel vectorsystem of the present disclosure is an expression vector systemcomprising one or more expression vectors, even more specifically arecombinant expression vector system comprising one or more recombinantexpression vectors. In various preferred embodiments, the vector type ofthe one or more vectors comprising the nucleic acid sequences encodingat least 4-1BBL, scIL-12 and IL-2 is the same for all vectors of thevector system. The vector typically is of a type that is capable ofcarrying the nucleic acid sequences encoding at least 4-1BBL, scIL-12and IL-2. Thus, the vector is of a type having the capacity of taking upthe nucleic acid sequences encoding at least 4-1BBL, scIL-12 and IL-2.Preferably, each of the one or more vectors comprising the nucleic acidsequences encoding at least 4-1BBL, scIL-12 and IL-2 is any one of anadenoviral vector, a retroviral vector, a lentiviral vector, a poxvirusvector, a vaccinia virus vector, preferably MVA, an adenovirus vector,an adenovirus-associated virus vector, a herpes virus vector, an alphavirus vector, and a measles virus vector. More preferably, each of theone or more vectors comprising the nucleic acid sequences encoding atleast 4-1BBL, scIL-12 and IL-2 is an adenoviral vector, a retroviralvector, a poxvirus vector, a vaccinia virus vector, preferably MVA, anadenovirus vector, an adenovirus-associated virus vector, or a herpesvirus vector. Still more preferably, each of the one or more vectorscomprising the nucleic acid sequences encoding at least 4-1BBL, scIL-12and IL-2 is an adenoviral vector, a herpes virus vector, or a vacciniavirus vector, preferably MVA.

The present disclosure demonstrates a relationship between IFN-γexpression and increasing 4-1BBL levels in the context of theimmunostimulating vector system that is based on one or more vectorscomprising nucleic acid sequences encoding at least 4-1BBL, scIL-12 andIL-2. As described in Example 11 and shown in FIG. 19, transgeneexpression of 4-1BBL, IL-2 and scIL-12 and IFN-γ response of Im02, andcombinations of single-gene expressing vectors reveal that IFN-γexpression is dependent on increasing 4-1BBL levels. Combinations ofconstant levels of IL-12 and IL-2, both at MOI [5], with increasinglevels of 4-1BBL up to MOI [100], lead to increasing IFN-γ induction atmoderate IL-12 levels, as shown in FIG. 19. The important finding of thepresent disclosure is that maximum induction of IFN-γ and the relatedimmune activation can be achieved not only by the specific arrangementof the nucleic acid sequences encoding 4-1BBL, scIL-12 and IL-2 asdepicted in vector Im02, but also by alternative embodiments thatprovide for an increased or higher expression of 4-1BBL as compared tothe expression of scIL-12 and IL-2. Thus, the present disclosureprovides a vector comprising nucleic acid sequences encoding 4-1BBL,scIL-12 and IL-2, wherein the vector provides for the production ofhigher or increased levels of 4-1BBL at constant levels of IL-12 andIL-2. More specifically, the present disclosure also provides a vectorcomprising nucleic acid sequences encoding 4-1BBL, scIL-12 and IL-2, andfurther comprising at least one regulatory nucleic acid sequenceproviding for an increased or higher expression level of 4-1BBL ascompared to the expression levels of scIL-12 and IL-2. This aspectreflects the finding that vector Im02 has been shown to provide for ahigher expression of 4-1BBL as compared to the expression of scIL-12 andIL-2, and that combinations of constant levels of IL-12 and IL-2, bothat MOI [5], with increasing levels of 4-1BBL up to MOI [100], lead toincreasing IFN-γ induction at moderate IL-12 levels, as discussed above.

FIGS. 3 and 19 show that the expression level of 4-1BBL obtained with avector or vector system of the present disclosure is increased ascompared to the expression level of 4-1BBL obtained by the expressionconstruct of vector Im01. Therefore, in a preferred embodiment, theexpression level of 4-1BBL is increased as compared to the expressionlevel of 4-1BBL obtained by the expression construct of vector Im01.

FIGS. 3 and 19 also show that the expression level of scIL-12 isdecreased as compared to the expression level of scIL-12 obtained by theexpression construct of vector Im01. Therefore, in a preferredembodiment, the expression level of scIL-12 is decreased as compared tothe expression level of scIL-12 obtained by the expression construct ofvector Im01.

FIGS. 3 and 19 further show that the expression level of IL-2 isincreased as compared to the expression level of IL-2 obtained by theexpression construct of vector Im01. Therefore, in a preferredembodiment, the expression level of IL-2 is increased as compared to theexpression level of IL-2 obtained by the expression construct of vectorIm01.

The present disclosure encompasses a vector comprising nucleic acidsequences encoding 4-1BBL, scIL-12 and IL-2, and further comprising atleast one regulatory nucleic acid sequence providing for an increased orhigher expression level of 4-1BBL as compared to the expression level of4-1BBL obtained by the expression construct of vector Im01. Preferably,the expression level of scIL-12 is decreased as compared to theexpression level of scIL-12 obtained by the expression construct ofvector Im01 and/or the expression level of IL-2 is increased as comparedto the expression level of IL-2 obtained by the expression construct ofvector Im01. More specifically, the at least one regulatory nucleic acidsequence provides for a decreased expression level of scIL-12 ascompared to the expression level of scIL-12 obtained by the expressionconstruct of vector Im01 and/or the at least one regulatory nucleic acidsequence provides for an increased expression level of IL-2 as comparedto the expression level of IL-2 obtained by the expression construct ofvector Im01.

The expression construct of vector Im01 is depicted in Example 1, andcomprises a CMV promoter, two IRES elements, scIL-12, 4-1BBL, IL-2 and apoly-A signal organized in 5′ to 3′ orientation in the followingsequential order: 5′-CMV-scIL-12-IRES-4-1BBL-IRES-IL-2-poly-A signal-3′.

More specifically, the expression construct of vector Im01 correspondsto the expression construct of the vector designated “Ad-3” shown inFIG. 1 of WO 2004/035799 A2. The expression construct of vector “Ad-3”is hereby incorporated into the present disclosure by reference. In FIG.1 of WO 2004/035799 A2, the designation “Ad-3” means the complete vectorcarrying the corresponding expression construct or cassette shown insaid FIG. 1. Accordingly, expression construct of vector “Ad-3” meansthe expression construct depicted in the bottom line of FIG. 1 of WO2004/035799 A2. In the present disclosure, the expression construct ofvector Im01 preferably corresponds to the expression construct of vector“Ad-3” as shown in FIG. 1 of WO 2004/035799 A2 and comprising human cDNAof the nucleic acid sequences encoding scIL-12, 4-1BBL, and IL-2.Accordingly, the expression construct of vector Im01 as referred to inthe present disclosure comprises the following elements organized in 5′to 3′ orientation in the following sequential order: 5′-CMV-humanscIL-12-PV-IRES-human 4-1BBL-EMCV-IRES-human IL-2-SV-40 poly-A-3′.

In various embodiments of the present disclosure, reference to“expression levels obtained by the expression construct of vector Im01”means “expression levels obtained by the vector Im01”. Vector Im01 is anadenoviral vector. Thus, in various embodiments of the presentdisclosure, reference to “expression levels obtained by the vector Im01”includes an adenoviral vector comprising the expression construct ofvector “Ad-3” as shown in FIG. 1 of WO 2004/035799 A2 with human cDNA ofthe nucleic acid sequences encoding scIL-12, 4-1BBL, and IL-2. Morespecifically, reference to “expression levels obtained by the vectorIm01” means expression levels obtained by an adenoviral vectorcomprising the following elements organized in 5′ to 3′ orientation inthe following sequential order: 5′-CMV-human scIL-12-PV-IRES-human4-1BBL-EMCV-IRES-human IL-2-SV-40 poly-A-3′. Thus, in preferredembodiments, reference to “expression levels obtained by the expressionconstruct of vector Im01” means “expression levels obtained by theexpression construct of vector Im01” as shown in FIG. 20. Accordingly,with reference to FIG. 20, vector Im01 means the shuttle vector hu pE1.1Im01. The expression construct of vector Im01 shown in FIG. 20encompasses the CMV promoter, human scIL-12, PV IRES, human 4-1BBL, EMCVIRES, human IL-2, and SV40polyA. The nucleotide sequence of the shuttlevector hu pE1.1 Im01 depicted in FIG. 20 has a total of 7,845 bp. Thenucleotide sequences of the CMV promoter, human scIL-12, PV IRES, human4-1BBL, EMCV IRES, human IL-2, and SV40polyA can be identified in thenucleotide sequence of SEQ ID NO: 11 (FIG. 17), which shows thenucleotide sequence of the shuttle vector hu pE1.1 Im02 depicted in FIG.2. The nucleotide sequences of the CMV promoter, human scIL-12, PV IRES,human 4-1BBL, EMCV IRES, human IL-2, and SV40polyA of the shuttle vectorhu pE1.1 Im01 correspond to the respective nucleotide sequences of theCMV promoter, human 4-1BBL, EMCV IRES, human IL-2, PV IRES, humanscIL-12, and SV40polyA of the shuttle vector hu pE1.1 Im02.

In various preferred embodiments, reference to “expression levelsobtained by the expression construct of vector Im01” means expressionlevels obtained by the expression construct of vector Im01 cloned orsub-cloned into a plasmid or vector carrying, e.g., adenoviral vectorDNA. More preferably, reference to “expression levels obtained by theexpression construct of vector Im01” means expression levels obtained bythe expression construct of vector Im01 cloned or sub-cloned into anadenoviral vector.

In various embodiments, the vector type is the same for the vectorcomprising the nucleic acid sequences encoding 4-1BBL, scIL-12 and IL-2,and the vector comprising the expression construct of vector Im01. Thevector typically is of a type that is capable of carrying the nucleicacid sequences encoding 4-1BBL, scIL-12 and IL-2. Thus, the vector is ofa type having the capacity of taking up the nucleic acid sequencesencoding 4-1BBL, scIL-12 and IL-2. Preferably, each of the one or morevectors comprising the nucleic acid sequences encoding 4-1BBL, scIL-12and IL-2 is any one of an adenoviral vector, a retroviral vector, alentiviral vector, a poxvirus vector, a vaccinia virus vector,preferably MVA, an adenovirus vector, an adenovirus-associated virusvector, a herpes virus vector, an alpha virus vector, and a measlesvirus vector. More preferably, each of the one or more vectorscomprising the nucleic acid sequences encoding 4-1BBL, scIL-12 and IL-2is an adenoviral vector, a retroviral vector, a poxvirus vector, avaccinia virus vector, preferably MVA, an adenovirus vector, anadenovirus-associated virus vector, or a herpes virus vector. Still morepreferably, each of the one or more vectors comprising the nucleic acidsequences encoding 4-1BBL, scIL-12 and IL-2 is an adenoviral vector, aherpes virus vector, or a vaccinia virus vector, preferably MVA.

In various embodiments of the present disclosure, reference to“expression levels obtained by the expression construct of vector Im01”or “expression levels obtained by the vector Im01” means expressionlevels obtained by application of a standard in vitro expression systemcomprising a tumor cell line, preferably human A549 cells, to betransduced with the vector (i.e., a vector or vector system of thepresent disclosure, or vector Im01 or a vector, preferably an adenoviralvector, comprising the expression construct of vector Im01), and asubsequent overlay of human blood immune cells as target cells.

Thus, the disclosure provides a vector comprising nucleic acid sequencesencoding 4-1BBL, scIL-12 and IL-2, and further comprising at least oneregulatory nucleic acid sequence providing for an increased expressionlevel of 4-1BBL as compared to the expression levels of scIL-12 andIL-2, wherein the expression levels are determined in an in vitroexpression system (or expression assay) comprising a tumor cell line,preferably a human A549 cell line, transduced with a vector, preferablyan adenoviral vector, comprising the expression construct of vectorIm01, and human blood immune cells, preferably peripheral bloodmononuclear cells (PBMCs).

In the present disclosure, the terms expression level or transgeneexpression level and expression rate or transgene expression rate may beused interchangeably.

The expression levels of 4-1BBL, IL-2 and scIL-12 provided by the vectoror vector system of the present disclosure induce an immune responsethat is transgene-specific, i.e., is specific for the expression levelsof 4-1BBL, IL-2 and scIL-12 provided by the vector or vector system ofthe present disclosure. The expression levels provided by the vector orvector system of the present disclosure induces an immune response thatis particularly specific for the increased expression level of 4-1BBL ascompared to the expression levels of scIL-12 and IL-2. Preferably, theexpression levels of 4-1BBL, IL-2 and scIL-12 provided by the vector orvector system of the present disclosure induce an immune response thatis particularly specific for the increased expression level of 4-1BBL ascompared to the expression levels of scIL-12 and IL-2, wherein theexpression level of 4-1BBL is increased as compared to the expressionlevel of 4-1BBL obtained by the expression construct of vector Im01.More preferably, the expression levels of 4-1BBL, IL-2 and scIL-12provided by the vector or vector system of the present disclosure inducean immune response that is particularly specific for the increasedexpression level of 4-1BBL as compared to the expression levels ofscIL-12 and IL-2, wherein the expression level of scIL-12 is decreasedand/or the expression level of IL-2 is increased as compared to theexpression levels of scIL-12 and/or IL-2 obtained by the expressionconstruct of vector Im01. Still more preferably, the expression levelsof 4-1BBL, IL-2 and scIL-12 provided by the vector or vector systems ofthe present disclosure induce an immune response that is particularlyspecific for the increased expression level of 4-1BBL as compared to theexpression levels of scIL-12 and IL-2, wherein the expression level of4-1BBL is increased as compared to the expression level of 4-1BBLobtained by the expression construct of vector Im01, and wherein theexpression level of scIL-12 is decreased and/or the expression level ofIL-2 is increased as compared to the expression levels of scIL-12 and/orIL-2 obtained by the expression construct of vector Im01.

The transgene-specific immune response preferably meanstransgene-specific IFN-γ response or transgene-specific expression orproduction of IFN-γ. Cytokine levels including IFN-γ levels canroutinely be assayed by ELISA.

In preferred embodiments, the transgene-specific immune response inducedby the expression levels of 4-1BBL, IL-2 and scIL-12 is detected ordetermined by an in vitro assay, preferably ELISA.

In various embodiments of the present disclosure, the nucleic acidsequences encoding 4-1BBL, IL-2 and scIL-12 are expressed by one vectorfrom two individual regulatory nucleic acid sequences, wherein oneregulatory nucleic acid sequence is located upstream of the nucleic acidsequence encoding 4-1BBL, and the other regulatory nucleic acid sequenceis located upstream of an expression cassette comprising nucleic acidsequences encoding IL-2 and scIL-12. Preferably, the said expressioncassette comprises nucleic acid sequences encoding IL-2 and scIL-12 in5′ to 3′ orientation in the order 5′-IL-2-scIL-12-3′. According to thepresent disclosure, the two regulatory nucleic acid sequences providefor an increased or higher expression level of 4-1BBL as compared to theexpression levels of scIL-12 and IL-2 and/or provide for an increased orhigher expression level of 4-1BBL as compared to the expression level of4-1BBL obtained by the expression construct of vector Im01. Preferably,the two regulatory nucleic acid sequences also or further provide for adecreased expression level of scIL-12 as compared to the expressionlevel of scIL-12 obtained by the expression construct of vector Im01and/or an increased expression level of IL-2 as compared to theexpression level of IL-2 obtained by the expression construct of vectorIm01. Preferably, the regulatory nucleic acid sequences are promotersequences.

In various embodiments of the present disclosure, the nucleic acidsequences encoding 4-1BBL, IL-2 and scIL-12 are expressed by one vectorfrom three individual regulatory nucleic acid sequences, wherein oneregulatory nucleic acid sequence is located upstream of the nucleic acidsequence encoding 4-1BBL, one regulatory nucleic acid sequence islocated upstream of the nucleic acid sequence encoding IL-2, and oneregulatory nucleic acid sequence is located upstream of the nucleic acidsequence encoding scIL-12. According to the present disclosure, thethree regulatory nucleic acid sequences provide for an increased orhigher expression level of 4-1BBL as compared to the expression levelsof scIL-12 and IL-2 and/or provide for an increased or higher expressionlevel of 4-1BBL as compared to the expression level of 4-1BBL obtainedby the expression construct of vector Im01. Preferably, the threeregulatory nucleic acid sequences also or further provide for adecreased expression level of scIL-12 as compared to the expressionlevel of scIL-12 obtained by the expression construct of vector Im01and/or an increased expression level of IL-2 as compared to theexpression level of IL-2 obtained by the expression construct of vectorIm01. Preferably, the regulatory nucleic acid sequences are promotersequences.

As described herein, regulatory nucleic acid sequence(s) preferablymeans promoter(s) (sequence(s)) or, more specifically, expressionpromoter(s) sequence(s)). Strong promoters that may be used for theintended increased or high expression of level of 4-1BBL include,without being limited thereto, any one of: Cytomegalovirus (CMV),Elongation Factor 1 Alpha (EF1A), Rous Sarcoma Virus (RSV), Simian Virus40 (SV40). CMV is a particularly preferred promoter. These promoters canbe used in various embodiments of the present disclosure, and representpreferred promoters that can provide for an increased expression levelof 4-1BBL as compared to the expression levels of scIL-12 and IL-2. Thisapplies in particular for embodiments, in which the three nucleic acidsequences encoding 4-1BBL, IL-2 and scIL-12 are organized in atricistronic cassette in 5′ to 3′ orientation in a sequential order 1,2, 3 with the nucleic acid sequence encoding 4-1BBL located upstream ofthe nucleic acid sequences encoding IL-2 and scIL-12, preferably whereinthe three nucleic acid sequences encoding 4-1BBL, IL-2 and scIL-12 areorganized from 5′ to 3′ in the following order:5′-4-1BBL-IL-2-scIL-12-3′.

Promoters that may be used for the intended expression of IL-2 and/orIL-12 include, without being limited thereto, Phosphoglycerate Kinase(PGK) and Ubiquitin C (UBC). These promoters can be used in variousembodiments of the present disclosure, and represent preferred promotersthat can provide for the intended expression level of IL-2 and IL-12. Inparticular, these promoters can be used in embodiments, in which thenucleic acid sequences encoding 4-1BBL, IL-2 and scIL-12 are expressedby one vector from two individual regulatory nucleic acid sequences,wherein one regulatory nucleic acid sequence is located upstream of thenucleic acid sequence encoding 4-1BBL, and the other regulatory nucleicacid sequence is located upstream of an expression cassette comprisingthe nucleic acid sequences encoding IL-2 and scIL-12. These promoterscan furthermore be used in embodiments, in which the nucleic acidsequences encoding 4-1BBL, IL-2 and scIL-12 are expressed by one vectorfrom three individual regulatory nucleic acid sequences, wherein oneregulatory nucleic acid sequence is located upstream of the nucleic acidsequence encoding 4-1BBL, one regulatory nucleic acid sequence islocated upstream of the nucleic acid sequence encoding IL-2, and oneregulatory nucleic acid sequence is located upstream of the nucleic acidsequence encoding scIL-12.

In various embodiments, the at least one regulatory nucleic acidsequence of the vector comprising nucleic acid sequences encoding4-1BBL, scIL-12 and IL-2, provides for the same expression level orexpression ratio of 4-1BBL, scIL-12 and IL-2 that is obtained with thevector Im02.

The one or more vectors of the novel vector/vector system of the presentdisclosure may be any one of an adenoviral vector, an adeno-associatedvirus vector, a lentiviral vector, a retroviral vector, a herpes simplexvirus vector, a pox virus vector, a RNA vector, a plasmid vector, ananoparticle vector, and naked DNA, or a combination thereof.

In various embodiments of the disclosure, the one or more vectors of thenovel vector/vector system are viral vectors. Viral vectors may be live,attenuated, replication-conditional or replication-deficient, and mayalso be a non-pathogenic (defective), replication-competent viralvector.

In certain embodiments of the present disclosure, the one or morevectors of the novel vector/vector system of the present disclosure areselected from retroviral vector genome, lentiviral vector genome,poxvirus vector genome, vaccinia virus vector genome, adenovirus vectorgenome, adenovirus-associated virus vector genome, herpes virus vectorgenome, alpha virus vector genome, plasmid DNA and RNA.

Safety features of the viral vector, e.g., integration deficiency, aredesirably incorporated. In certain embodiments integration deficiencymay be conferred by elements of the vector genome but may also derivefrom elements of the packaging system (e.g., a non functional integraseprotein that may not be part of the vector genome but supplied intrans).

In various embodiments, the one or more vectors of the novelvector/vector system of the disclosure are replicative vectors.Preferably, the replicative vectors are replicating viral vectors, morepreferably replicating adenoviral vectors. In various other embodiments,the one or more vectors of the novel vector/vector system of the presentdisclosure are non-replicative vectors, preferably non-replicationcompetent viral vectors, more preferably non-replication competentadenoviral vectors.

If the one or more vectors of the novel vector/vector system of thedisclosure are RNA vectors, these may comprise inserted modifiedribonucleotides.

Exemplary viral vectors of the present disclosure include, but are notlimited to, retroviral vectors, lentiviral vectors, poxvirus vectors,vaccinia virus vectors, adenovirus vectors, adenovirus-associated virusvectors, herpes virus vectors, and alpha virus vectors. Preferably, theviral vector is an adenoviral vector.

In certain embodiments, an adenovirus vector or adenovirus-associatedvirus vector may be used for expressing the three genes 4-1BB ligand(4-1BBL), single chain IL-12 (scIL-12) and IL-2. Several adenovirusvector systems and methods for administering the vectors have beendescribed (see, e.g., Mercier et al., Proc. Natl. Acad. Sci. USA, 2004,101:6188-93). Retroviral vectors may include those based upon murineleukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropicretroviruses, simian immunodeficiency virus (SIV), humanimmunodeficiency virus (HIV), and combinations thereof.

In certain embodiments, the one or more vectors of the novelvector/vector system of the disclosure is a retroviral vector,preferably a lentiviral vector. Suitable genome-based lentiviral vectorsfor human gene therapy include those based on Human ImmunodeficiencyVirus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equineinfectious anemia virus, Vesicular Stomatitis Virus (VSV), and SimianImmunodeficiency Virus (SIV).

In various embodiments, the vector is plasmid DNA or cosmid DNA. PlasmidDNA or cosmid DNA containing one or more polynucleotides encoding atleast 4-1BBL, scIL-12 and IL-2 as described herein are readilyconstructed using standard techniques well known in the art. The vectormay be typically constructed in a plasmid form that can then betransfected into a packaging or producer cell line. The plasmidgenerally comprises sequences useful for replication of the plasmid inbacteria. Such plasmids are well known in the art. In addition, vectorsthat include a prokaryotic origin of replication may also include a genewhose expression confers a detectable or selectable marker such as adrug resistance.

In one embodiment, recombinant expression vectors are providedcomprising a polynucleotide sequence encoding at least 4-1BBL, scIL-12and IL-2 that induce an immune response in an infectious disease orcancer setting. For directing expression of 4-1BBL, scIL-12 and IL-2,the encoding polynucleotide sequences in each vector should include atleast one appropriate expression control sequence (also called aregulatory expression sequence or feature) that is operatively linked tothe encoding polynucleotide sequence(s). Expression control elementsthat may be used for regulating the expression of the encodedpolypeptides are known in the art and include, but are not limited to,inducible promoters, constitutive promoters, secretion signals,enhancers, leaders and other regulatory sequences.

As described herein, the expression vector may comprise at least oneregulatory expression sequence (expression control sequence). In certainembodiments, when the expression vector comprises a viral vector genome,expression of 4-1BBL, scIL-12 and IL-2 is desired in particular targetcells. Typically, for example, in a viral vector the polynucleotidesequence encoding 4-1BBL, scIL-12 and/or IL-2 is located between the 5′LTR and 3′ LTR sequences. Furthermore, the encoding nucleotidesequence(s) is preferably operatively linked in a functionalrelationship with other genetic or regulatory sequences or features, forexample transcription regulatory sequences including promoters orenhancers, that regulate expression of the genes encoding 4-1BBL,scIL-12 and IL-2 in a particular manner. With respect to viral vectorconstructs, an “internal” promoter/enhancer is one that is locatedbetween the 5′ LTR and the 3′ LTR sequences in the viral vector and isoperatively linked to the encoding polynucleotide sequence of interest.The internal promoter/enhancer may be any promoter, enhancer orpromoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship. A “functionalrelationship” and “operatively linked” mean, without limitation, thatthe sequence is in the correct location and orientation with respect tothe promoter and/or enhancer such that the sequence of interest will beexpressed when the promoter and/or enhancer is contacted with theappropriate molecules. In certain instances, the useful transcriptionalregulatory sequences are those that are highly regulated with respect toactivity, both temporally and spatially. The choice of an internalpromoter/enhancer is based on the desired expression pattern of thethree genes 4-1BBL, scIL-12 and IL-2 and the specific properties ofknown promoters/enhancers. Thus, the internal promoter may beconstitutively active. Non-limiting examples of constitutive promotersthat may be used include the CMV promoter. In various embodiments of thedisclosure, the novel vector/vector system comprises an internalpromoter/enhancer, which provides for a higher expression of 4-1BBL ascompared to scIL-12 and IL-2. Many enhancers in viral genomes and inmammalian genomes have been identified and characterized (see, e.g.,publically available databases such as GenBank).

Enhancers are typically cis-acting elements of DNA, usually about 10 to300 bp in length, that act on a promoter to increase its transcription.Many enhancer sequences are now known from mammalian genes and fromeukaryotic cell viruses. Examples include the SV40 enhancer on the lateside of the replication origin (bp 100-270), the cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. The enhancer may bespliced into the vector at a position 5′ or 3′ to the polynucleotidesequence encoding the gene(s) of interest, but is preferably located ata site 5′ from the promoter. An enhancer can be used in combination witha heterologous promoter. One of ordinary skill in the art will be ableto select the appropriate enhancer based on the desired expressionpattern of 4-1BBL, scIL-12 and IL-2.

In various embodiments, the promoter may be a tissue specific promoter.In some embodiments, the promoter is a target cell-specific promoter. Inaddition, promoters may be selected to allow for inducible expression of4-1BBL, scIL-12 and/or IL-2. A number of systems for inducibleexpression are known in the art, including the tetracycline responsivesystem, the lac operator-repressor system, as well as promotersresponsive to a variety of environmental or physiological changes,including heat shock, metal ions, interferons, hypoxia, steroids, andradiation. A combination of promoters may also be used to obtain thedesired expression of each of the three genes encoding 4-1BBL, scIL-12and IL-2. The artisan of ordinary skill will be able to select apromoter based on the desired expression pattern of the polynucleotidesequence(s) in the organism or the target tissue or target cell ofinterest.

As described herein, the expression vector, including a viral vectorgenome, may comprise at least one RNA Polymerase II or III responsivepromoter. This promoter can be operatively linked to the polynucleotidesequence(s) encoding at least 4-1BBL, scIL-12 and/or IL-2 and can alsobe linked to a termination sequence. In addition, more than one RNAPolymerase II or III promoter may be incorporated. RNA polymerase II andIII promoters are well known to the person of skill in the art.

When targeting delivery of a recombinant expression vector, including aviral vector genome, to a particular target cell or target tissue forinducing a cell-mediated immune response, the vector genome will usuallycontain a promoter that is recognized by the target cell or targettissue and that is operatively linked to the sequence(s) of interest,viral components (when the vector is a viral vector), and othersequences discussed herein.

Promoters may be inducible, constitutive, temporally active or tissuespecific. Inducible promoters are useful tools in genetic engineeringbecause the expression of genes to which they are operatively linked canbe turned on or off, e.g., in a particular tissue. Inducible promoterscan be grouped as chemically-regulated promoters, andphysically-regulated promoters. Typical chemically-regulated promotersinclude, not are not limited to, steroid-regulated promoter (e.g., ratglucocorticoid receptor (GR)-based promoter, human estrogen receptor(ER)-based promoter), metal-regulated promoters (e.g., metallothioneingene-based promoters), and pathogenesis-related promoters (e.g.,Arabidopsis and maize pathogen-related (PR) protein-based promoters).Typical physically-regulated promoters include, but are not limited to,temperature-regulated promoters (e.g., heat shock promoters), andlight-regulated promoters (e.g., soybean SSU promoter). Other exemplarypromoters are well known to the person skilled in the art.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat can be used. The use of other viral or mammaliancellular or bacterial phage promoters, which are well-known in the artto achieve expression of polynucleotides, is contemplated as well. Theone of skill in the art will be able to select an appropriate promoterbased on the specific circumstances. Many different promoters aredescribed in the art, as are methods for operatively linking thepromoter to the polynucleotide sequence to be expressed. Both nativepromoter sequences and many heterologous promoters may be used to directexpression in a packaging cell and a target cell or target tissue.Heterologous promoters are typically used because they generally permitgreater transcription and higher yields of the desired protein ascompared to the native promoter. The promoter may be obtained, forexample, from the genomes of viruses such as polyoma virus, fowlpoxvirus, adenovirus, bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40). The promoter may also be, for example, a heterologous mammalianpromoter, for example, the actin promoter or an immunoglobulin promoter,a heat-shock promoter, or the promoter normally associated with thenative sequence, provided such promoters are compatible with the targetcell or target tissue. In one embodiment, the promoter is the naturallyoccurring viral promoter in a viral expression system. In someembodiments, the promoter is a tumor cell-specific promoter. Expressionvectors may also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. These sequences are oftenfound in the 5′ and, occasionally 3′, untranslated regions of eukaryoticor viral DNAs or cDNAs and are well known in the art. The novelvector/vector system of the disclosure may further encode one or moreimmunogens, which include, but are not limited to, immunogens from anoncogenic virus (e.g., EBV, HPV, HBV, HCV, HTLV, and KSHV) andtumor-associated antigens. Preferably, the tumor associated antigen is atumor-associated antigen from bladder cancer, liver cancer, Merkel cellcarcinoma, renal cell carcinoma, prostate cancer, mesothelioma,pancreatic cancer, melanoma, breast cancer, colorectal cancer, lungcancer, ovarian cancer. More preferably, the tumor-associated antigen isfrom bladder cancer. In certain embodiments, the tumor associatedantigen is any one of p53, Ras, c-Myc, A-Raf, B-Raf, C-Raf, NY-ESO-1,LAGE-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12,CT7, CT10, GAGE, IMP3, BK T-antigen, MART-1, DAM-6, NA88-A, Gp100, PSA,PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT,MUC1, MUC2, TRK receptors, PRAME, P15, SART-1, SART-2, SART-3, Wilms'tumor antigen (WT1), AFP, CEA, ELF2M, GnT-V, G250, HSP70-2M, HST-2,MUM-1, MUM-2, MUM-3, RAGE, 707-AP, BCR-ABL, interferon regulatory factor4 (IRF4), Tumor-associated calcium signal transducer 1 (TACSTD1)TACSTD2, receptor tyrosine kinases, Epidermal Growth Factor Receptor(EGFR), platelet derived growth factor receptor (PDGFR), vascularendothelial growth factor receptor (VEGFR), cytoplasmic tyrosinekinases, src-family, Nuclear Factor-Kappa B (NF-κB), Notch receptors,c-Met, extracellular signal-regulated kinases (ERKs), PMSA, PR-3, MDM2,Mesothelin, renal cell carcinoma-5T4, SM22-alpha, STEAD, hTERT, sarcomatranslocation breakpoints, EpCAM, NA17, PAX3, ALK, androgen receptor,cyclin B1, MYCN, BORIS, sperm protein 17, SSX2, B7H3, TIE2, Page4,MAD-CT-1, FAP, MAD-CT-2, and fos related antigen 1. In anotherembodiment, a method is provided herein wherein the tumor-associatedantigen is selected from a bladder cancer antigen. In one embodiment,the bladder cancer antigen is any one of CTA, NY-ESO-1, LAGE-1, MAGE-A1,MAGE-A3, MAGE-A4, MAGE-A10, CT7, CT10, GAGE, PRAME; BAGE; RAGE, SAGE,HAGE, MPHOSPH1, DEPDCI, IMP3 and MAGE-A, and BK T-antigen.

When the expression vector is a viral vector genome, the viral vectorgenome may be typically constructed in a plasmid form that may betransfected into a packaging or producer cell line for production of theviral vector genome construct. The plasmid generally comprises sequencesuseful for replication of the plasmid in bacteria. Such plasmids arewell known in the art. In addition, vectors that include a prokaryoticorigin of replication may also include a gene whose expression confers adetectable or selectable marker such as a drug resistance. As will beunderstood by those of skill in the art, it can be advantageous tomodify a coding sequence to enhance its expression in a particular host.The genetic code is redundant with 64 possible codons, but mostorganisms preferentially use a subset of these codons. The codons thatare utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias”. Optimized coding sequencescontaining codons preferred by a particular prokaryotic or eukaryotichost can be prepared, for example, to increase the rate of translationor to produce recombinant RNA transcripts having desirable properties,such as a longer half-life, as compared with transcripts produced from anon-optimized sequence. In various embodiments, the nucleic acidsequence(s) encoding at least 4-1BBL, scIL-12, and/or IL-2 iscodon-optimized for expression in human. In preferred embodiments of thepresent disclosure, the nucleic acid sequence encoding 4-1BBL is humancDNA (human gene of 4-1BBL), the nucleic acid sequence encoding scIL-12is human cDNA (human gene of scIL-12), and/or the nucleic acid sequenceencoding IL-2 is human cDNA (human gene of IL-2).

In various embodiments, the nucleic acid sequence(s) of the novelvector/vector system of the present disclosure are cDNA sequences. Thenucleic acid sequences of the genes of 4-1BBL, scIL-12, and/or IL-2contained in the novel vector/vector system of the present disclosuremay be considered as heterologous nucleic acid sequences. The termsnucleic acid sequence(s) and polynucleotide(s) may be usedinterchangeably herein. As described herein, the term “nucleic acidsequence” may encompass both single-stranded and double-stranded nucleicacid sequences. In various embodiments, a nucleic acid sequence is DNAsequence.

In various embodiments, the vector is a DNA vector. In variousembodiments, the vector is an RNA vector. The novel vector of thepresent disclosure may be more specifically described as a circularvector, even more specifically as a circular expression vector. Asdescribed herein, the term “vector” may encompass both single-strandedand double-stranded vectors, including, but not limited to,single-stranded and double-stranded DNA vectors.

As described herein, nucleic acid sequences of genes encoding 4-1BBLencompass nucleic acid sequences encoding variants of 4-1BBL, inparticular variants of 4-1BBL having the amino acid sequence as shown inSEQ ID NO: 2 (FIG. 13). Such variant sequences are described in furtherdetail herein below. Likewise, nucleic acid sequences of genes encodingIL-2 encompass nucleic acid sequences encoding variants of IL-2, inparticular variants of IL-2 having the amino acid sequence as shown inSEQ ID NO: 4 (FIG. 14). Such variant sequences are described in furtherdetail herein below. Also, nucleic acid sequences of genes encodingscIL-12 encompass nucleic acid sequences encoding variants of scIL-12,in particular variants of scIL-12 having the amino acid sequence asshown in SEQ ID NO: 6 (FIG. 15). Such variant sequences are described infurther detail herein below.

In various embodiments of the disclosure, the nucleic acid sequence (ofthe gene) encoding 4-1BBL comprises (i) the nucleic acid sequence of SEQID NO: 1, wherein the nucleic acid sequence is codon-optimized forexpression in human, (ii) the nucleic acid sequence (of the gene)encoding IL-2 comprises the nucleic acid sequence of SEQ ID NO: 3,wherein the nucleic acid sequence is codon-optimized for expression inhuman, and/or (iii) the nucleic acid sequence (of the gene) encodingscIL-12 comprises the nucleic acid sequence of SEQ ID NO: 5, wherein thenucleic acid sequence is codon-optimized for expression in human.

4-1BB is a member of the Tumor Necrosis Factor (TNF) receptor family.CD137 is the designation for 4-1BB according to the CD nomenclature. Inthe present disclosure, the terms CD137 and 4-1BB can be usedinterchangeably. Accordingly, the terms 4-1BB ligand (4-1BBL) and CD137ligand can also be used interchangeably in the present disclosure. Invarious embodiments of the disclosure, the nucleic acid sequenceencoding 4-1BBL (or CD137 ligand) shows at least 70% homology orsequence identity to the nucleic acid sequence of SEQ ID NO: 1 (FIG.13), wherein the variant nucleic acid sequence encodes a 4-1BBL proteincapable of specifically binding activated T cells, preferably CD4+helper cells and CD8+ T cells. Preferably, the nucleic acid sequenceencoding 4-1BBL shows at least 80% homology or sequence identity to thenucleic acid sequence of SEQ ID NO: 1 (FIG. 13), wherein the variantnucleic acid sequence encodes a 4-1BBL protein capable of specificallybinding activated T cells, preferably CD4+ helper cells and CD8+ Tcells. More preferably, the nucleic acid sequence encoding 4-1BBL showsat least 90% homology or sequence identity to the nucleic acid sequenceof SEQ ID NO: 1 (FIG. 13), wherein the variant nucleic acid sequenceencodes a 4-1BBL protein capable of specifically binding activated Tcells, preferably CD4+ helper cells and CD8+ T cells. Even morepreferably, the nucleic acid sequence encoding 4-1BBL shows at least95%, 96%, 97%, 98%, or 99% homology or sequence identity to the nucleicacid sequence of SEQ ID NO: 1 (FIG. 13), wherein the variant nucleicacid sequence encodes a 4-1BBL protein capable of specifically bindingactivated T cells, preferably CD4+ helper cells and CD8+ T cells. Incertain embodiments, variants of 4-1BBL as described above exhibit thesame binding specificity for T cells, preferably CD4+ helper cells andCD8+ T cells, as the native 4-1BBL encoded by the sequence of SEQ ID NO:1 (FIG. 13). In a particularly preferred embodiment, the nucleic acidsequence encoding 4-1BBL comprises (or consists of) the sequence of SEQID NO: 1 (FIG. 13). In various preferred embodiments, a variant sequenceof the nucleic acid sequence of human 4-1BBL as described herein is nota nucleotide sequence encoding mouse 4-1BBL. Thus, in various preferredembodiments the novel vector/vector system of the present disclosuredoes not comprise a nucleic acid sequence encoding 4-1BBL of mouseorigin.

In various embodiments of the disclosure the nucleic acid sequenceencoding IL-2 shows at least 70% homology or sequence identity to thenucleic acid sequence of SEQ ID NO: 3 (FIG. 14), wherein the variantnucleic acid sequence encodes an IL-2 protein having immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity.Preferably, the nucleic acid sequence encoding IL-2 shows at least 80%homology or sequence identity to the nucleic acid sequence of SEQ ID NO:3 (FIG. 14), wherein the variant nucleic acid sequence encodes an IL-2protein having immune stimulating activity, preferably T helper cell andCD8+ T cell stimulating activity. More preferably, the nucleic acidsequence encoding IL-2 shows at least 90% homology or sequence identityto the nucleic acid sequence of SEQ ID NO: 3 (FIG. 14), wherein thevariant nucleic acid sequence encodes an IL-2 protein having immunestimulating activity, preferably T helper cell and CD8+ T cellstimulating activity. Even more preferably, the nucleic acid sequenceencoding IL-2 shows at least 95%, 96%, 97%, 98% or 99% homology orsequence identity to the nucleic acid sequence of SEQ ID NO: 3 (FIG.14), wherein the variant nucleic acid sequence encodes an IL-2 proteinhaving immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity. In certain embodiments, variants of IL-2 asdescribed above exhibit the same immune stimulating activity, preferablyT helper cell and CD8+ T cell stimulating activity, as the native IL-2encoded by the sequence of SEQ ID NO: 3 (FIG. 14). In a particularlypreferred embodiment, the nucleic acid sequence encoding IL-2 comprises(or consists of) the sequence of SEQ ID NO: 3 (FIG. 14). In variouspreferred embodiments, a variant sequence of the nucleic acid sequenceof human IL-2 as described herein is not a nucleotide sequence encodingmouse IL-2. Thus, in various preferred embodiments the novelvector/vector system of the present disclosure does not comprise anucleic acid sequence encoding IL-2 of mouse origin.

In various embodiments of the disclosure, the nucleic acid sequenceencoding scIL-12 shows at least 70% homology or sequence identity to thenucleic acid sequence of SEQ ID NO: 5 (FIG. 15), wherein the variantnucleic acid sequence encodes a scIL-12 protein having immunestimulating activity, preferably T helper cell and CD8+ T cellstimulating activity. Preferably, the nucleic acid sequence encodingscIL-12 shows at least 80% homology or sequence identity to the nucleicacid sequence of SEQ ID NO: 5 (FIG. 15), wherein the variant nucleicacid sequence encodes a scIL-12 protein having immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity.More preferably, the nucleic acid sequence encoding scIL-12 shows atleast 90% homology or sequence identity to the nucleic acid sequence ofSEQ ID NO: 5 (FIG. 15), wherein the variant nucleic acid sequenceencodes a scIL-12 protein having immune stimulating activity, preferablyT helper cell and CD8+ T cell stimulating activity. Even morepreferably, the nucleic acid sequence encoding scIL-12 shows at least95%, 96%, 97%, 98% or 99% homology or sequence identity to the nucleicacid sequence of SEQ ID NO: 5 (FIG. 15), wherein the variant nucleicacid sequence encodes a scIL-12 protein having immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity.As described herein, a protein is considered a scIL-12 protein if itcomprises an amino acid sequence comprising the two subunits of thenative IL-12 protein as a fusion protein. The sequence of SEQ ID NO: 5(FIG. 15) shows the sequence of the gene encoding the 40 kDa and 35 kDasubunits of human IL-12 linked by a linker. In the nucleotide sequenceof SEQ ID NO: 5 depicted in FIG. 15, the linker sequence is shown inboldface. In certain embodiments, variants of scIL-12 as described aboveexhibit the same immune stimulating activity, preferably T helper celland CD8+ T cell stimulating activity, as the native scIL-12 encoded bythe sequence of SEQ ID NO: 5 (FIG. 15). In a particularly preferredembodiment, the nucleic acid sequence encoding scIL-12 comprises (orconsists of) the sequence of SEQ ID NO: 5 (FIG. 15). In variouspreferred embodiments, a variant sequence of the nucleic acid sequenceof human scIL-12 as described herein is not a nucleotide sequenceencoding mouse scIL-12. Thus, in various preferred embodiments the novelvector/vector system of the present disclosure does not comprise anucleic acid sequence encoding scIL-12 of mouse origin.

In various embodiments of the disclosure, the nucleic acid sequence of(the gene for) 4-1BBL encodes a 4-1BBL polypeptide comprising an aminoacid sequence having at least 70% homology or sequence identity to theamino acid sequence of SEQ ID NO: 2 (FIG. 13), wherein the 4-1BBLpolypeptide is capable of specifically binding activated T cells,preferably activated CD4+ T helper cells and CD8+ T cells. Preferably,the nucleic acid sequence of (the gene for) 4-1BBL encodes a 4-1BBLpolypeptide comprising an amino acid sequence having at least 80%homology or sequence identity to the amino acid sequence of SEQ ID NO: 2(FIG. 13), wherein the 4-1BBL polypeptide is capable of specificallybinding T cells, preferably activated CD4+ T helper cells and CD8+ Tcells. More preferably, the nucleic acid sequence of (the gene for)4-1BBL encodes a 4-1BBL polypeptide comprising an amino acid sequencehaving at least 90% homology or sequence identity to the amino acidsequence of SEQ ID NO: 2 (FIG. 13), wherein the 4-1BBL polypeptide iscapable of specifically binding activated T cells, preferably CD8+ Tcells. Even more preferably, the nucleic acid sequence of (the gene for)4-1BBL encodes a 4-1BBL polypeptide comprising an amino acid sequencehaving at least 95%, 96%, 97%, 98%, or 99% homology or sequence identityto the amino acid sequence of SEQ ID NO: 2 (FIG. 13), wherein the 4-1BBLpolypeptide is capable of specifically binding activated T cells,preferably activated CD4+ T helper cells and CD8+ T cells. In certainembodiments, variant nucleic acid sequences of (the gene for) 4-1BBL asdescribed above encode a 4-1BBL polypeptides, which exhibit the samebinding specificity for T cells, preferably for CD8+ T cells, as thenative 4-1BBL having the amino acid sequence of SEQ ID NO: 2 (FIG. 13).In a preferred embodiment, the nucleic acid sequence of (the gene for)4-1BBL encodes a polypeptide comprising (or consisting of) the aminoacid sequence of SEQ ID NO: 2 (FIG. 13).

In various embodiments of the disclosure, the nucleic acid sequence of(the gene for) IL-2 encodes an IL-2 polypeptide comprising an amino acidsequence having at least 70% homology or sequence identity to the aminoacid sequence of SEQ ID NO: 4 (FIG. 14), wherein the IL-2 polypeptidehas immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity. Preferably, the nucleic acid sequence of (thegene for) IL-2 encodes an IL-2 polypeptide comprising an amino acidsequence having at least 80% homology or sequence identity to the aminoacid sequence of SEQ ID NO: 4 (FIG. 14), wherein the IL-2 polypeptidehas immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity. More preferably, the nucleic acid sequence of(the gene for) IL-2 encodes an IL-2 polypeptide comprising an amino acidsequence having at least 90% homology or sequence identity to the aminoacid sequence of SEQ ID NO: 4 (FIG. 14), wherein the IL-2 polypeptidehas immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity. Even more preferably, the nucleic acidsequence of (the gene for) IL-2 encodes an IL-2 polypeptide comprisingan amino acid sequence having at least 95%, 96%, 97%, 98% or 99%homology or sequence identity to the amino acid sequence of SEQ ID NO: 4(FIG. 14), wherein the IL-2 polypeptide has immune stimulating activity,preferably T helper cell and CD8+ T cell stimulating activity. Incertain embodiments, variant nucleic acid sequences of IL-2 as describedabove encode IL-2 polypeptides, which exhibit the same immunestimulating activity, preferably T helper cell and CD8+ T cellstimulating activity, as the native IL-2 having the amino acid sequenceof SEQ ID NO: 4 (FIG. 14). In a preferred embodiment, the nucleic acidsequence of (the gene for) IL-2 encodes a polypeptide comprising (orconsisting of) the amino acid sequence of SEQ ID NO: 4 (FIG. 14).

In various embodiments of the disclosure, the nucleic acid sequence of(the gene for) scIL-12 encodes a scIL-12 polypeptide comprising an aminoacid sequence having at least 70% homology or sequence identity to theamino acid sequence of SEQ ID NO: 6 (FIG. 15), wherein the scIL-12polypeptide has immune stimulating activity, preferably monocyte, Thelper cell and CD8+ T cell stimulating activity. Preferably, thenucleic acid sequence of (the gene for) scIL-12 encodes a scIL-12polypeptide comprising an amino acid sequence having at least 80%homology or sequence identity to the amino acid sequence of SEQ ID NO: 6(FIG. 15), wherein the scIL-12 polypeptide has immune stimulatingactivity, preferably monocyte, T helper cell and CD8+ T cell stimulatingactivity. More preferably, the nucleic acid sequence of (the gene for)scIL-12 encodes a scIL-12 polypeptide comprising an amino acid sequencehaving at least 90% homology or sequence identity to the amino acidsequence of SEQ ID NO: 6 (FIG. 15), wherein the scIL-12 polypeptide hasimmune stimulating activity, preferably monocyte, T helper cell and CD8+T cell stimulating activity. Even more preferably, the nucleic acidsequence of (the gene for) scIL-12 encodes a scIL-12 polypeptidecomprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or99% homology or sequence identity to the amino acid sequence of SEQ IDNO: 6 (FIG. 15), wherein the scIL-12 polypeptide has immune stimulatingactivity, preferably monocyte, T helper cell and CD8+ T cell stimulatingactivity. In certain embodiments, variant nucleic acid sequences ofscIL-12 as described above encode scIL-12 polypeptides, which exhibitthe same immune stimulating activity, preferably T helper cell and CD8+T cell stimulating activity, as the native scIL-12 having the amino acidsequence of SEQ ID NO: 6 (FIG. 15). In a preferred embodiment, thenucleic acid sequence of (the gene for) scIL-12 encodes a polypeptidecomprising (or consisting of) the amino acid sequence of SEQ ID NO: 6(FIG. 15).

As described herein, one or more poly- or multicistronic expressionunits may be used that include two or three polynucleotide sequencesencoding two or all three proteins, i.e., two polynucleotide sequenceseach encoding at least (i) 4-1BBL or scIL-12, or (ii) 4-1BBL or IL-2, or(iii) scIL-12 or IL-2; or three polynucleotide sequences each encodingat least 4-1BBL, scIL-12, or 4-1BBL, respectively. The use ofmulticistronic vectors (or expression units) reduces the total number ofnucleic acid molecules required and thus may avoid possible difficultiesassociated with coordinating expression from multiple vector genomes. Ina multicistronic vector the various elements to be expressed may beoperatively linked to one or more promoters (and other expressioncontrol elements as necessary).

When using several promoters, one may observe mutual inhibition of thepromoters. Particularly high expression rates can be achieved usingvectors that are at least tricistronic, and which are furthercharacterized in that they contain only one promoter per expressioncassette, and furthermore comprise an IRES sequence for each cistronthat is not localized immediately downstream of the promoter. Thecombination of promoters and IRES (internal ribosomal entry sites)sequences is considered to provide for an improved protein expression.The use of different IRES sequences may provide for the additionaladvantage that the frequency of recombination can be among thesesequences can be minimized. In various embodiments of the presentdisclosure, the IRES is from EMCV (encephalomyocarditis virus). Invarious embodiments of the present disclosure, the IRES is from PV(poliovirus). In various embodiments of the disclosure, the novelvector/vector system comprises two IRES between the three transgenes4-1BBL (CD137L), IL-2 and single chain IL-12, wherein one IRES may bethe EMCV IRES, and the other IRES may be the PV IRES. In variousembodiments of the disclosure, the novel vector/vector system comprisestwo IRES between the three transgenes 4-1BBL (CD137L), IL-2 and singlechain IL-12, wherein both IRES may be the EMCV IRES, or both IRES may bethe PV IRES. When using tetracistronic vectors, it may be useful tosplit them onto different expression cassettes. In this case, it ispreferred that one promoter is present per expression cassette. Theseparation in two expression cassettes, which preferably show themaximum possible distance from each other, provides for spatiallyseparating the promoters, thereby reducing mutual inhibition.

In various embodiments, the novel vector/vector system of the presentdisclosure comprises the three genes of 4-1BBL (CD137L), IL-2 and singlechain IL-12 organized in this order (i.e., 5′ to 3′ with the geneencoding IL-2 located downstream of the gene encoding 4-1BBL, and thegene encoding scIL-12 located downstream of the gene encoding IL-2) in atricistronic construct linked by internal ribosomal entry sites (IRES).Preferably, the three genes are genes of human 4-1BBL (CD137L), humanIL-2 and human single chain IL-12. More preferably, the three genes aredriven by a Cytomegalovirus (CMV) promoter, i.e., the vector systemcomprises a CMV promoter upstream of the tricistronic constructcomprising the three genes of human 4-1BBL (CD137L), human IL-2 andhuman single chain IL-12. Still more preferably, transcriptpolyadenylation is induced by a SV40-derived signal, i.e., the novelvector/vector system comprises a gene encoding a SV40 polyadenylationsignal downstream of the tricistronic construct comprising the threegenes of human 4-1BBL (CD137L), human IL-2 and human single chain IL-12(human scIL-12). Even more preferably, the vector DNA is adenoviralvector DNA.

Thus, in one preferred embodiment, the novel vector/vector system of thepresent disclosure comprises an expression cassette comprising (i) thethree genes of human 4-1BBL (CD137L), human IL-2 and human single chainIL-12 organized in this order (i.e., 5′ to 3′ with the gene encodingIL-2 located downstream of the gene encoding 4-1BBL, and the geneencoding scIL-12 located downstream of the gene encoding IL-2) in atricistronic construct linked by internal ribosomal entry sites (IRES),(ii) a CMV promoter upstream of the tricistronic construct comprisingthe three genes of human 4-1BBL (CD137L), human IL-2 and human singlechain IL-12, and (iii) a gene encoding a SV40 polyadenylation signaldownstream of the said tricistronic construct, wherein the vector is anadenoviral vector.

In various embodiments, the novel vector/vector system of the disclosurecomprises an expression construct, in which the three genes of 4-1BBL(CD137L), IL-2 and single chain IL-12 are organized as depicted in anyone of (a) to (f) below (Prom.=Promoter):

(a) -Prom.>-|hu 4-1BBL|-|hu IL-2|-|hu scIL-12|-(b) -Prom.>-|hu 4-1BBL|-(IRES)-|hu IL-2|-(IRES)-|hu scIL-12|-(c) -Prom.>-|hu 4-1BBL|-(IRES)-|hu IL-2|-(IRES)-|hu scIL-12|-|polyA|-(d) -Prom.>-|hu 4-1BBL|-Prom.>-|hu IL-2|-|hu scIL-12|-(e) -Prom.>-|hu 4-1BBL|-|hu IL-2-Prom.>-|hu scIL-12|-(f) -Prom.>-|hu 4-1BBL|-Prom.>-|hu IL-2-Prom.>-|hu scIL-12|-

In various embodiments, the novel vector of the present disclosurecomprises a promoter upstream of (an expression construct comprising)the three genes of 4-1BBL, IL-2 and scIL-12 organized in an order asdescribed elsewhere herein. Preferably, the gene of human 4-1BBL is atposition 1 of the three genes of the novel vector, more specifically atposition 1 of the expression construct comprising the said three genes.

In various embodiments, the novel vector of the present disclosurecomprises an expression cassette comprising a nucleic acid sequencehaving at least 70% homology or sequence identity to the nucleic acidsequence of SEQ ID NO: 12 (FIG. 18), wherein expression of the variantnucleic acid sequence provides for the same immune stimulating activity,preferably T helper cell and CD8+ T cell stimulating activity, as theexpression of the expression cassette of SEQ ID NO: 12. Preferably, theexpression cassette comprises a nucleic acid sequence having at least80% homology or sequence identity to the nucleic acid sequence of SEQ IDNO: 12 (FIG. 18), wherein expression of the variant nucleic acidsequence provides for the same immune stimulating activity, preferably Thelper cell and CD8+ T cell stimulating activity, as the expression ofthe expression cassette of SEQ ID NO: 12. More preferably, theexpression cassette comprises a nucleic acid sequence having at least90% homology or sequence identity to the nucleic acid sequence of SEQ IDNO: 12 (FIG. 18), wherein expression of the variant nucleic acidsequence provides for the same immune stimulating activity, preferably Thelper cell and CD8+ T cell stimulating activity, as the expression ofthe expression cassette of SEQ ID NO: 12. Even more preferably, theexpression cassette comprises a nucleic acid sequence having at least95%, 96%, 97%, 98% or 99% homology or sequence identity to the nucleicacid sequence of SEQ ID NO: 12 (FIG. 18), wherein expression of thevariant nucleic acid sequence provides for the same immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity,as the expression of the expression cassette of SEQ ID NO: 12. In apreferred embodiment, the novel vector of the present disclosurecomprises an expression cassette comprising (or consisting of) thenucleic acid sequence of SEQ ID NO: 12 (FIG. 18).

In various embodiments, the novel vector of the present disclosurecomprises an expression cassette comprising a nucleic acid sequencehaving at least 70% homology or sequence identity to a referenceexpression cassette comprising the following nucleic acid sequences ofSEQ ID NO: 11 (FIG. 17): (i) the nucleic acid sequence of bp 1,080-1,844(human 4-1BBL), (ii) the nucleic acid sequence of bp 1,885-2,388 (EMCVIRES), (iii) the nucleic acid sequence of bp 2,409-2,870 (human IL-2),(iv) the nucleic acid sequence of bp 2,914-3,545 (PV IRES), and (v) thenucleic acid sequence of bp 3,581-5,203 (human scIL-12 comprising p40and p35 subunit of human IL-12 linked by a linker), wherein expressionof the variant expression cassette provides for the same immunestimulating activity, preferably T helper cell and CD8+ T cellstimulating activity, as the expression of the said reference expressioncassette. Preferably, the expression cassette comprises a nucleic acidsequence having at least 80% homology or sequence identity to the saidreference expression cassette, wherein expression of the variantexpression cassette provides for the same immune stimulating activity,preferably T helper cell and CD8+ T cell stimulating activity, as theexpression of the said reference expression cassette. More preferably,the expression cassette comprises a nucleic acid sequence having atleast 90% homology or sequence identity to the said reference expressioncassette, wherein expression of the variant expression cassette providesfor the same immune stimulating activity, preferably T helper cell andCD8+ T cell stimulating activity, as the expression of the saidreference expression cassette. Even more preferably, the expressioncassette comprises a nucleic acid sequence having at least 95%, 96%,97%, 98% or 99% homology or sequence identity to the said referenceexpression cassette, wherein expression of the variant expressioncassette provides for the same immune stimulating activity, preferably Thelper cell and CD8+ T cell stimulating activity, as the expression ofthe said reference expression cassette. In a preferred embodiment, thenovel vector of the present disclosure comprises an expression cassettecomprising (or consisting of) the following nucleic acid sequences ofSEQ ID NO: 11 (FIG. 17): (i) the nucleic acid sequence of bp 1,080-1,844(human 4-1BBL), (ii) the nucleic acid sequence of bp 1,885-2,388 (EMCVIRES), (iii) the nucleic acid sequence of bp 2,409-2,870 (human IL-2),(iv) the nucleic acid sequence of bp 2,914-3,545 (PV IRES), and (v) thenucleic acid sequence of bp 3,581-5,203 (human scIL-12 comprising p40and p35 subunit of human IL-12 linked by a linker).

In various embodiments, the novel vector of the present disclosurecomprises an expression cassette comprising a nucleic acid sequencehaving at least 70% homology or sequence identity to a referenceexpression cassette comprising the following nucleic acid sequences ofSEQ ID NO: 11 (FIG. 17): (i) the nucleic acid sequence of bp 484-1,059(CMV promoter), (ii) the nucleic acid sequence of bp 1,080-1,844 (human4-1BBL), (iii) the nucleic acid sequence of bp 1,885-2,388 (EMCV IRES),(iv) the nucleic acid sequence of bp 2,409-2,870 (human IL-2), (v) thenucleic acid sequence of bp 2,914-3,545 (PV IRES), (vi) the nucleic acidsequence of bp 3,581-5,203 (human scIL-12 comprising p40 and p35 subunitof human IL-12 linked by a linker), and (vii) the nucleic acid sequenceof bp 5,271-5,510 (SVpolyA), wherein expression of the variantexpression cassette provides for the same immune stimulating activity,preferably T helper cell and CD8+ T cell stimulating activity, as theexpression of the said reference expression cassette. Preferably, theexpression cassette comprises a nucleic acid sequence having at least80% homology or sequence identity to the said reference expressioncassette, wherein expression of the variant expression cassette providesfor the same immune stimulating activity, preferably T helper cell andCD8+ T cell stimulating activity, as the expression of the saidreference expression cassette. More preferably, the expression cassettecomprises a nucleic acid sequence having at least 90% homology orsequence identity to the said reference expression cassette, whereinexpression of the variant expression cassette provides for the sameimmune stimulating activity, preferably T helper cell and CD8+ T cellstimulating activity, as the expression of the said reference expressioncassette. Even more preferably, the expression cassette comprises anucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% homologyor sequence identity to the said reference expression cassette, whereinexpression of the variant expression cassette provides for the sameimmune stimulating activity, preferably T helper cell and CD8+ T cellstimulating activity, as the expression of the said reference expressioncassette. In a preferred embodiment, the novel vector of the presentdisclosure comprises an expression cassette comprising (or consistingof) the following nucleic acid sequences of SEQ ID NO: 11 (FIG. 17): (i)the nucleic acid sequence of bp 484-1,059 (CMV promoter), (ii) thenucleic acid sequence of bp 1,080-1,844 (human 4-1BBL), (iii) thenucleic acid sequence of bp 1,885-2,388 (EMCV IRES), (iv) the nucleicacid sequence of bp 2,409-2,870 (human IL-2), (v) the nucleic acidsequence of bp 2,914-3,545 (PV IRES), (vi) the nucleic acid sequence ofbp 3,581-5,203 (human scIL-12 comprising p40 and p35 subunit of humanIL-12 linked by a linker), and (vii) the nucleic acid sequence of bp5,271-5,510 (SVpolyA).

In various embodiments, the vector system comprises the three genes of4-1BBL (CD137L), IL-2 and single chain IL-12 organized in three separatevectors. Preferably, the three genes are human genes of 4-1BBL (CD137L),IL-2 and single chain IL-12. More preferably, the three genes are eachdriven by a Cytomegalovirus (CMV) promoter, i.e., each vector comprisesa CMV promoter upstream of the gene of human 4-1BBL (CD137L), human IL-2and human single chain IL-12, respectively. Still more preferably,transcript polyadenylation is induced by a SV40-derived signal, i.e.,each vector system comprises a gene encoding a SV40 polyadenylationsignal downstream of the gene of human 4-1BBL (CD137L), human IL-2 andhuman single chain IL-12 (human scIL-12), respectively. Even morepreferably, the vector DNA of each separate vector is adenoviral vectorDNA.

Thus, in one preferred embodiment, the vector system comprises threeadenoviral vectors, each comprising (i) the gene of human 4-1BBL(CD137L), human IL-2 or human single chain IL-12, (ii) a CMV promoterupstream of the gene of human 4-1BBL (CD137L), human IL-2 and humansingle chain IL-12, respectively, and (iii) a nucleic acid sequenceencoding a SV40 polyadenylation signal downstream of the gene of human4-1BBL (CD137L), human IL-2 and human single chain IL-12, respectively.

In various aspects, the vector system comprises the three genes of4-1BBL (CD137L), IL-2 and single chain IL-12 organized in two separatevectors. Specifically, in various embodiments, one of the two vectorscomprises the genes of 4-1BBL (CD137L) and IL-2, and the other vectorcomprises the gene of single chain IL-12. Preferably, the genes arehuman genes of 4-1BBL (CD137L), IL-2 and single chain IL-12. Morepreferably, the genes are driven by a Cytomegalovirus (CMV) promoter,i.e., the vector comprising the genes of human 4-1BBL (CD137L) and humanIL-2 comprises a CMV promoter upstream of the gene of human 4-1BBL(CD137L) in case the gene of human 4-1BBL (CD137L) is located upstreamof the gene of human IL-2, or upstream of the gene of human IL-2 in casethe gene of human IL-2 is located upstream of the gene of human 4-1BBL(CD137L), and the vector comprising the gene of human scIL-12 comprisesa CMV promoter upstream of the gene of human scIL-12. Still morepreferably, transcript polyadenylation is induced by a SV40-derivedsignal, i.e., each of the two vectors comprises a nucleic acid sequenceencoding a SV40 polyadenylation signal, i.e., the vector comprising thegenes of human 4-1BBL (CD137L) and human IL-2 comprises a gene encodinga SV40 polyadenylation signal downstream of the gene of human IL-2 incase the gene of human 4-1BBL (CD137L) is located upstream of the geneof human IL-2, or comprises a gene encoding a SV40 polyadenylationsignal downstream of the gene of human 4-1BBL (CD137L) in case the geneof human IL-2 is located upstream of the gene of human 4-1BBL (CD137L),and the vector comprising the gene of human scIL-12 comprises a nucleicacid sequence encoding a SV40 polyadenylation signal downstream of thegene of human scIL-12. Even more preferably, the vector DNA of eachseparate vector is adenoviral vector DNA.

Thus, in one preferred embodiment, the vector system comprises twoadenoviral vectors, one comprising the genes of human 4-1BBL (CD137L)and human IL-2, and the other comprising the gene of human scIL-12,wherein (i) the vector comprising the genes of human 4-1BBL (CD137L) andhuman IL-2 further comprises a CMV promoter upstream of the gene ofhuman 4-1BBL (CD137L) in case the gene of human 4-1BBL (CD137L) islocated upstream of the gene of human IL-2, or upstream of the gene ofhuman IL-2 in case the gene of human IL-2 is located upstream of thegene of human 4-1BBL (CD137L), and wherein the vector comprising thegene of human scIL-12 comprises a CMV promoter upstream of the gene ofhuman scIL-12, and (ii) the vector comprising the genes of human 4-1BBL(CD137L) and human IL-2 still further comprises a gene encoding a SV40polyadenylation signal downstream of the gene of human IL-2 in case thenucleic acid sequence of human 4-1BBL (CD137L) is located upstream ofthe gene of human IL-2, or a gene encoding a SV40 polyadenylation signaldownstream of the gene of human 4-1BBL (CD137L) in case the gene ofhuman IL-2 is located upstream of the gene of human 4-1BBL (CD137L), andwherein the vector comprising the gene of human scIL-12 comprises a geneencoding a SV40 polyadenylation signal downstream of the gene of humanscIL-12.

In various other embodiments, the vector system comprises two separatevectors, wherein one of the two vectors comprises the genes of 4-1BBL(CD137L) and single chain IL-12 (scIL-12), and the other vectorcomprises the gene of IL-2. Preferably, the genes are human genes of4-1BBL (CD137L), scIL-12 and IL-2. More preferably, the genes are drivenby a Cytomegalovirus (CMV) promoter, i.e., the vector comprising thegenes of human 4-1BBL (CD137L) and human scIL-12 comprises a CMVpromoter upstream of the gene of human 4-1BBL (CD137L) in case the geneof human 4-1BBL (CD137L) is located upstream of the gene of humanscIL-12, or upstream of the gene of human scIL-12 in case the gene ofhuman scIL-12 is located upstream of the gene of human 4-1BBL (CD137L),and the vector comprising the gene of human IL-2 comprises a CMVpromoter upstream of the gene of human IL-2. Still more preferably,transcript polyadenylation is induced by a SV40-derived signal, i.e.,each of the two vectors comprises a nucleic acid sequence encoding aSV40 polyadenylation signal, i.e., the vector comprising the genes ofhuman 4-1BBL (CD137L) and human scIL-12 comprises a gene encoding a SV40polyadenylation signal downstream of the gene of human scIL-12 in casethe gene of human 4-1BBL (CD137L) is located upstream of the gene ofhuman scIL-12, or comprises a gene encoding a SV40 polyadenylationsignal downstream of the gene of human 4-1BBL (CD137L) in case the geneof human scIL-12 is located upstream of the gene of human 4-1BBL(CD137L), and the vector comprising the gene of human IL-2 comprises anucleic acid sequence encoding a SV40 polyadenylation signal downstreamof the gene of human IL-2. Even more preferably, the vector DNA of eachseparate vector is adenoviral vector DNA.

Thus, in one preferred embodiment, the vector system comprises twoadenoviral vectors, one comprising the genes of human 4-1BBL (CD137L)and human scIL-12, and the other comprising the gene of human IL-2,wherein (i) the vector comprising the genes of human 4-1BBL (CD137L) andhuman scIL-12 further comprises a CMV promoter upstream of the gene ofhuman 4-1BBL (CD137L) in case the gene of human 4-1BBL (CD137L) islocated upstream of the gene of human scIL-12, or upstream of the geneof human scIL-12 in case the gene of human scIL-12 is located upstreamof the gene of human 4-1BBL (CD137L), and wherein the vector comprisingthe gene of human IL-2 comprises a CMV promoter upstream of the gene ofhuman IL-2, and (ii) the vector comprising the genes of human 4-1BBL(CD137L) and human scIL-12 still further comprises a gene encoding aSV40 polyadenylation signal downstream of the gene of human scIL-12 incase the gene of human 4-1BBL (CD137L) is located upstream of the geneof human scIL-12, or a nucleic acid sequence encoding a SV40polyadenylation signal downstream of the gene of human 4-1BBL (CD137L)in case the gene of human scIL-12 is located upstream of the gene ofhuman 4-1BBL (CD137L), and wherein the vector comprising the gene ofhuman IL-2 comprises a nucleic acid sequence encoding a SV40polyadenylation signal downstream of the gene of human IL-2.

In various other embodiments, the vector system comprises two separatevectors, wherein one of the two vectors comprises the genes of IL-2 andsingle chain IL-12 (scIL-12), and the other vector comprises the gene of4-1BBL (CD137L). Preferably, the genes are human genes of IL-2, scIL-12and 4-1BBL (CD137L). More preferably, the genes are driven by aCytomegalovirus (CMV) promoter, i.e., the vector comprising the genes ofhuman IL-2 and human scIL-12 comprises a CMV promoter upstream of thegene of human IL-2 in case the gene of human IL-2 is located upstream ofthe gene of human scIL-12, or upstream of the gene of human scIL-12 incase the gene of human scIL-12 is located upstream of the gene of humanIL-2, and the vector comprising the gene of human 4-1BBL (CD137L)comprises a CMV promoter upstream of the gene of human 4-1BBL (CD137L).Still more preferably, transcript polyadenylation is induced by aSV40-derived signal, i.e., each of the two vectors comprises a nucleicacid sequence encoding a SV40 polyadenylation signal, i.e., the vectorcomprising the genes of human IL-2 and human scIL-12 comprises a nucleicacid sequence encoding a SV40 polyadenylation signal downstream of thegene of human scIL-12 in case the gene of human IL-2 is located upstreamof the gene of human scIL-12, or comprises a nucleic acid sequenceencoding a SV40 polyadenylation signal downstream of the gene of humanIL-2 in case the gene of human scIL-12 is located upstream of the geneof human IL-2, and the vector comprising the gene of human 4-1BBL(CD137L) comprises a nucleic acid sequence encoding a SV40polyadenylation signal downstream of the gene of human 4-1BBL (CD137L).Even more preferably, the vector DNA of each separate vector isadenoviral vector DNA.

Thus, in one preferred embodiment, the vector system comprises twoadenoviral vectors, one comprising the genes of human IL-2 and humanscIL-12, and the other comprising the gene of human 4-1BBL (CD137L),wherein (i) the vector comprising the genes of human IL-2 and humanscIL-12 further comprises a CMV promoter upstream of the gene of humanIL-2 in case the gene of human IL-2 is located upstream of the gene ofhuman scIL-12, or upstream of the gene of human scIL-12 in case the geneof human scIL-12 is located upstream of the gene of human IL-2, andwherein the vector comprising the gene of human 4-1BBL (CD137L)comprises a CMV promoter upstream of the gene of human 4-1BBL (CD137L),and (ii) the vector comprising the genes of human IL-2 and human scIL-12still further comprises a gene encoding a SV40 polyadenylation signaldownstream of the gene of human scIL-12 in case the gene of human IL-2is located upstream of the gene of human scIL-12, or a nucleic acidsequence encoding a SV40 polyadenylation signal downstream of the geneof human IL-2 in case the gene of human scIL-12 is located upstream ofthe gene of human IL-2, and wherein the vector comprising the gene ofhuman 4-1BBL (CD137L) comprises a nucleic acid sequence encoding a SV40polyadenylation signal downstream of the gene of human 4-1BBL (CD137L).For virus particles of the disclosure, the expression units furtherinclude a sequence encoding an envelope/capsid molecule or one or morematuration factors necessary for production of the desired vectorparticle in packaging cells.

Internal ribosome entry sites (IRES) elements are used to createmultigene, or multi- or polycistronic, messages. IRES elements are ableto bypass the ribosome scanning model of 5′ methylated Cap dependenttranslation and begin translation at internal sites. IRES elements canbe linked to heterologous open reading frames. Multiple open readingframes can be transcribed together, each separated by an IRES, creatingmulti- or polycistronic messages. Each component to be expressed in amulticistronic expression vector may be separated by an IRES element toallow for separate expression of the various proteins from the samepromoter. Tools that can be used to separate genetic elements in amulticistronic vector, in particular IRES elements, are known in theart. The efficacy of a particular multicistronic vector can readily betested by detecting expression of each of the genes using standardprotocols. In various embodiments of the present disclosure, the nucleicacid sequence(s) encoding at least 4-1BBL, scIL-12 and IL-2 areorganized in one vector, wherein the nucleic acid sequence(s) encodingscIL-12 and IL-2 are located downstream of the nucleic acid sequenceencoding 4-1BBL. Such a vector is considered a multicistronic expressionvector. In certain embodiments, the multicistronic expression vector isa tricistronic expression vector containing the nucleic acid sequences(of the genes) encoding 4-1BBL, scIL-12 and IL-2, wherein the genes areorganized in 5′ to 3′ orientation in a sequential order 1, 2, 3, withthe proviso that the gene encoding scIL-12 is not at position 1. Invarious embodiments, the nucleic acid sequence(s) encoding scIL-12 andIL-2 are located downstream of the nucleic acid sequence encoding4-1BBL. Preferably, in a multicistronic or tricistronic expressionvector, the nucleic acid sequence encoding IL-2 is located downstream ofthe nucleic acid sequence encoding 4-1BBL, and the nucleic acid sequenceencoding scIL-12 is located downstream of the nucleic acid sequenceencoding IL-2. In various embodiments of a multicistronic ortricistronic expression, a promoter is located upstream of the nucleicacid sequence encoding 4-1BBL, but not upstream of the nucleic acidsequence(s) encoding scIL-12 and/or IL-2.

In certain embodiments of a multicistronic or tricistronic expressionvector, the nucleic acid sequence(s) encoding at least 4-1BBL, scIL-12and/or IL-2 are linked by internal ribosomal entry sites (I RES).

In a specific exemplification, a viral vector genome comprises: anenhancer/promoter sequence, preferably a cytomegalovirus (CMV)enhancer/promoter sequence; the R and U5 sequences from a virus 5′ LTR;optionally a packaging sequence; an internal enhancer; an internalpromoter; one or more polynucleotides encoding at least 4-1BBL, scIL-12and/or IL-12; a U3 element with a deletion of its enhancer sequence; andthe R and U5 sequences of a viral 3′ LTR. Construction of the vectorgenome can be accomplished using any suitable genetic engineeringtechniques known in the art, including, without limitation, the standardtechniques of restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example asdescribed, e.g., in Sambrook et al. (1989 and 2001 editions; MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY).Vectors constructed for transient expression in mammalian cells may alsobe used. Transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of a the polypeptide encoded by thepolynucleotide in the expression vector. See Sambrook et al., supra, pp.16.17-16.22, 1989. Other vectors and methods suitable for adaptation tothe expression of polypeptides are well known in the art and are readilyadapted to the specific circumstances.

By using the teachings provided herein and the knowledge in the art, aperson skilled in the art will recognize that the efficacy of aparticular expression system can be tested by transfecting packagingcells with a vector comprising a polynucleotide sequence encoding areporter protein and measuring the expression using a suitabletechnique, for example, measuring fluorescence from a green fluorescentprotein conjugate. Other suitable reporter genes are well known in theart.

It is also contemplated by the present disclosure that in certainembodiments the novel vector/vector system provided by the presentdisclosure may comprise native IL-12 instead of scIL-12, i.e., maycomprise a nucleic acid sequence (of a gene) encoding native IL-12.IL-12 is a disulfide-linked heterodimeric cytokine composed of the twoseparately encoded p35 and p40 subunits. In various embodiments, thenucleic acid sequence (of a gene) may encode native human IL-12.Accordingly, in various embodiments, the novel vector/vector systemprovided by the present disclosure may comprise three genes encoding (i)4-1BB ligand (4-1BBL), (ii) IL-12 p40 and p35 subunits, and (iii) IL-2,wherein the said genes are organized in 5′ to 3′ orientation in asequential order 1, 2, and 3 with the proviso that the gene(s) encodingthe IL-12 p35 and IL-12 p40 subunits is/are not at position 1.

In various embodiments, the novel vector/vector system of the presentdisclosure may comprise three genes encoding 4-1BB ligand (4-1BBL),native IL-12, and IL-2, wherein the said genes are organized in 5′ to 3′orientation in a sequential order 1, 2, and 3, with the proviso that thegene(s) encoding the IL-12 p35 and p40 subunits is/are not at position1, wherein the gene(s) encoding IL-12 subunits p40 and p35 may comprisea nucleic acid sequence having at least 70% homology or sequenceidentity to the nucleic acid sequences of SEQ ID NOs: 7 and 9 (FIG. 16),wherein the variant nucleic acids sequence encodes an IL-12 proteinhaving immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity. Preferably, the gene(s) encoding IL-12subunits p35 and p40 may comprise a nucleic acid sequence having atleast 80% homology or sequence identity to the nucleic acid sequences ofSEQ ID NOs: 7 and 9 (FIG. 16), wherein the variant nucleic acidssequence encodes an IL-12 protein having immune stimulating activity,preferably T helper cell and CD8+ T cell stimulating activity. Morepreferably, the gene(s) encoding IL-12 subunits p40 and p35 may comprisea nucleic acid sequence having at least 90% homology or sequenceidentity to the nucleic acid sequences of SEQ ID NOs: 7 and 9 (FIG. 16),wherein the variant nucleic acids sequence encodes an IL-12 proteinhaving immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity. Even more preferably, the gene(s) encodingIL-12 subunits p40 and p35 may comprise a nucleic acid sequence havingat least 95%, 96%, 97%, 98% or 99% homology or sequence identity to thenucleic acid sequences of SEQ ID NOs: 7 and 9 (FIG. 16), wherein thevariant nucleic acid sequence encodes an IL-12 protein having immunestimulating activity, preferably T helper cell and CD8+ T cellstimulating activity. In particularly preferred embodiments, the gene(s)encoding IL-12 subunits p40 and p35 may comprise the nucleic acidsequences of SEQ ID NOs: 7 and 9 (FIG. 16). In certain embodiments,variants of IL-12 subunits p35 and p40 as described above exhibit thesame immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity, as the IL-12 subunits p35 and p40 encoded bythe sequences of SEQ ID NOs: 7 and 9.

In various embodiments, the gene(s) of IL-12 subunits p40 and p35 encodea polypeptide having at least 70% homology or sequence identity to theamino acid sequences of SEQ ID NOs: 8 and 10 (FIG. 16), wherein thepolypeptide has immune stimulating activity, preferably T helper celland CD8+ T cell stimulating activity. Preferably, the gene(s) of IL-12subunits p40 and p35 encode(s) a polypeptide having at least 70%homology or sequence identity to the amino acid sequences of SEQ ID NOs:8 and 10 (FIG. 16), wherein the polypeptide has immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity.More preferably, the gene(s) of IL-12 subunits p40 and p35 encode(s) apolypeptide having at least 90% or 95% homology or sequence identity tothe amino acid sequences of SEQ ID NOs: 8 and 10 (FIG. 16), wherein thepolypeptide has immune stimulating activity, preferably T helper celland CD8+ T cell stimulating activity. Even more preferably, the gene(s)of IL-12 subunits p40 and p35 encode(s) a polypeptide having the aminoacid sequences of SEQ ID NOs: 8 and 10 (FIG. 16).

Preferably, a variant sequence of the nucleic acid sequence of humanIL-12 subunits p35 and p40 as described herein is not a nucleotidesequence encoding the mouse IL-12 subunits p35 and p40. Thus, preferablythe novel vector/vector system of the present disclosure comprisingthree genes encoding 4-1BB ligand (4-1BBL), IL-12 subunits p35 and p40,and IL-2 as described above (i.e., wherein the said genes are organizedin 5′ to 3′ orientation in a sequential order 1, 2, and 3, with theproviso that the gene(s) encoding IL-12 subunits p35 and p40 is/are notat position 1), does not comprise a nucleic acid sequence encoding IL-12subunits p35 and p40 of mouse origin.

In another embodiment, vector particles are provided. The presentdisclosure provides virus particles comprising a vector system of thepresent disclosure. A vector particle comprises any one of the novelvector/vector systems described herein that comprise one or more vectorscomprising polynucleotide sequence(s) encoding at least 4-1BBL, scIL-12and/or IL-2. Also provided herein are methods for delivering a vectorsystem of the disclosure encoding at least 4-1BBL, scIL-12 and/or IL-2(as described herein) to a target cell. Such methods comprise contacting(i.e., permitting interaction) of the target cell with a vehicle thatdelivers the vector system of the disclosure. In particular embodiments,methods for delivering the novel vector/vector system comprisecontacting the cell by administering to a subject a vector particle thatcomprises a novel vector/vector system of the disclosure that thatcomprises one or more vectors comprising polynucleotide sequence(s)encoding at least 4-1BBL, scIL-12 and/or IL-2. In certain embodiments,the vector particle is a viral vector particle and the one or morevectors of the novel vector/vector system is any of a RNA vector, aplasmid vector, a nanoparticle vector, and naked DNA. In other certainembodiments, the vector particle is a particle derived from bacteriasuch as, for example, Listeria monocytogenes, Salmonella spp.,Mycobacterium bovis, Escherichia coli, Shigella spp., and Yersinia spp.,and the one or more vectors of the vector system is any of a RNA vector,a plasmid vector, a nanoparticle vector, and naked DNA. Exemplary viralvector particles include a lentiviral vector particle that comprises alentiviral vector genome; a poxvirus vector particle that comprises apoxvirus vector genome; a vaccinia virus vector particle that comprisesa vaccinia virus vector genome; an adenovirus vector particle thatcomprises a adenovirus vector genome; an adenovirus-associated virusvector particle that comprises a adenovirus-associated virus vectorgenome; a herpes virus vector particle that comprises a herpes virusvector genome (e.g., Herpes simplex virus I or II); or an alpha virusvector particle that comprises an alpha virus vector genome.

The vector particles (e.g., the viral vector particles described herein)may be injected in vivo, in particular to a tumor, where the particlesprovide for an immunostimulating effect by expression of 4-1BBL,scIL-12, and IL-2. The amount of viral particles is at least 3×10⁶ ivp(infectious viral particles), and can be at least 1)(10⁷ ivp, at least3×10⁷ ivp, at least 1)(10⁸ ivp, at least 3×10⁸ ivp, at least 1)(10⁹ ivp,or at least 3×10⁹ ivp. At selected intervals, cells from the recipient'smalignant (tumor) or target pathogen-infected tissue may be used tomeasure expression of 4-1BBL, scIL-12 and IL-2, for example, byobserving marker expression, such as GFP or luciferase if co-expressedby a polynucleotide sequence present in the vector system included inthe vector particle. In particular, T-cells from malignant (tumor) ortarget pathogen-infected tissue of vector particle-treated recipientsmay be measured for expression of 4-1BBL, scIL-12 and IL-2.

The term “replication competent adenoviral vector” refers to anyadenoviral vector that is not deficient in any gene function requiredfor viral replication in specific cells or tissues. The vector must iscapable of replicating and being packaged, but might replicate onlyconditionally in specific cells or tissues.

Adenovirus (Ad) is a large (about 36 kb) DNA virus that infects humans,but which displays a broad host range. There are approximately 50serotypes of human adenovirus, which are divided into six families basedon molecular, immunological, and functional criteria. By adulthood,virtually every human has been infected with the more common adenovirusserotypes, the major effect being cold-like symptoms. Adenoviralinfection of host cells results in adenoviral DNA being maintainedepisomally, which reduces the potential genotoxicity associated withintegrating vectors. Also, adenoviruses are structurally stable, and nogenome rearrangement has been detected after extensive amplification.

The adenoviral vectors used in the present disclosure can be anyadenoviral vectors suitable for use in a method of treating a human oranimal. Alternatively, various types of adenoviral vectors can be usedaccording to the present disclosure. Also, the vectors may be modifiedin any way known in the art, e.g. by deleting, inserting, mutating ormodifying any viral areas. The vectors can be made tumor specific withregard to replication. For example, the adenoviral vector may comprisemodifications in E1, E3 and/or E4 such as the insertion of tumorspecific promoters, deletions of areas and insertion of transgenes.

Human Ad-5 is a human adenovirus serotype that is well characterizedgenetically and biochemically (GenBank M73260; AC_000008). Thus, in apreferred embodiment, the adenovirus is a replication competent Ad5serotype or a hybrid serotype comprising an Ad5 component. Theadenovirus may be a wild type strain or may be genetically modified toenhance tumor selectivity, for example by attenuating the ability of thevirus to replicate within normal quiescent cells without affecting theability of the virus to replicate in tumor cells. Non-limiting examplesof adenoviruses encompassed by the present disclosure include Delta-24,Delta-24-RGD, ICOVIR-5, ICOVIR-7, ONYX-015, ColoAd1, and H101. In oneparticular embodiment, the adenovirus is Delta-24 or Delta-24-RGD. TheDelta-24 adenovirus is derived from adenovirus type 5 (Ad-5) andcontains a 24-base-pair deletion within the CR2 portion of the E1 Agene. Delta-24-RGD further comprises an insertion of the RGD-4Csequence. The E1 A deletion increases the selectivity of the virus forcancer cells; the RGD-4C sequence increases the infectivity of the virusin gliomas.

Furthermore, the backbone of the adenoviral vector may be of anyserotype. Still further, the vectors may be chimeric vectors, e.g. Ad5/3vectors. As an example, “Ad5/3 vector” refers to a chimeric vectorhaving parts of both Ad5 and Ad3 vectors.

The adenovirus is an attractive delivery system, and is well-establishedfor use in gene transfer in several therapeutic applications. Theadenovirus enters the permissive host cell via a cell surface receptor,and it is then internalized.

The absence or the presence of low levels of the coxsackie virus andadenovirus receptor (CAR) on tumor types can limit the efficacy of theadenovirus. Modifying the capsid allows CAR independent target cellinfection. Also, adenoviral uptake into target tissue can be improved bythe addition of transfectant-like polycationic compounds. It has beendemonstrated herein (see Example 10) that the adenoviral uptake isparticularly improved using the transfectant protamine sulfate. Thus, invarious embodiments, protamine sulfate is used for transfection of thevector systems of the present disclosure, preferably for transfection ofan adenoviral vector system of the disclosure.

In expression, one will typically include a polyadenylation signal toeffect proper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the present disclosure, and any such sequence may beemployed. Preferred embodiments include the SV40 polyadenylation signaland/or the bovine growth hormone polyadenylation signal, convenientand/or known to function well in various target cells. Also contemplatedherein as an element of the expression construct is a transcriptionaltermination site. These elements can serve to enhance message levelsand/or to minimize read through from the cassette into other sequences.

In certain embodiments of the disclosure, cells infected by the novelvector/vector system of the disclosure may be identified in vitro byincluding a reporter gene in the vector system. Generally, a selectablereporter is one that confers a property that allows for selection. Apositive selectable reporter is one in which the presence of thereporter gene allows for its selection, while a negative selectablereporter is one in which its presence prevents its selection. An exampleof a positive selectable marker is a drug resistance marker. Other typesof reporters include screenable reporters such as GFP (green fluorescentprotein).

Embodiments of the disclosure can use current viral vector platformtechnologies designed to create vaccines or gene therapy constructs.Aspects of the viral vector construction include inserting geneticmaterial into a viral vector and confirming the construct throughcharacterization and sequencing of the nucleic acid, virus and virusproduct. The viral vector is then put through a series of feasibilitiesstudies designed to assess scalability.

The present disclosure provides a polynucleotide comprising a nucleicacid sequence encoding the novel vector/vector system of the disclosure.

Furthermore, the present disclosure provides a composition comprisingthe novel vector/vector system or a virus particle as disclosed herein.The present disclosure also provides a composition comprising apolynucleotide disclosed herein, i.e., a polynucleotide comprising anucleic acid sequence encoding the novel vector/vector system of thedisclosure.

Without being bound by theory, the use of the novel vector/vector systemexpressing 4-1BBL, scIL-12 and IL-2 results in the local production ofcytokines in or around the tumor, which will direct systemically orlocally induced tumor specific cytotoxic T-cells to the site of thecancer. Through the immune-stimulatory effect provided by thismechanism, the efficacy of immunotherapy of cancers, viral infectionsand immune system disorders will be greatly enhanced. Theimmunostimulating effect provided by the novel vector/vector system ofthe present disclosure has been demonstrated in the examples.

The present disclosure encompasses compositions comprising the novelvector/vector system or virus particle disclosed herein for use as amedicament. The present disclosure provides a medicament comprising thenovel vector/vector system or a virus particle disclosed herein. Thepresent disclosure also provides a medicament comprising apolynucleotide disclosed herein, i.e., a polynucleotide comprising anucleic acid sequence encoding the novel vector/vector system of thedisclosure.

The present disclosure encompasses the novel vector/vector system orvirus particle disclosed herein for use as a therapeutic vaccine, morespecifically for use as a therapeutic vaccine for the treatment ofcancer or viral infections.

The present disclosure also provides an immune cell or a cancer celltransduced or transfected with the novel vector/vector system or a virusparticle disclosed herein. Such transduced or transfected cells may beused for ex vivo therapies, in particular ex vivo cancer therapy. Invarious embodiments of such therapies, at least 5% of the cancer cellstransduced or transfected with the novel vector/vector system or a virusparticle disclosed herein are expressing 4-1BBL.

In the present disclosure, the cancer cell preferably is a cell of atumor, i.e., a tumor cell, more specifically a cell of a solid tumor.

Furthermore, the present disclosure provides a composition comprisingsuch an immune cell or cancer cell transduced or transfected with thenovel vector/vector system or a virus particle disclosed herein. Stillfurther, the present disclosure provides a medicament comprising such animmune cell or cancer cell transduced or transfected with the novelvector/vector system or a virus particle disclosed herein. In variousembodiments, the immune cell is a T-cell, an NK cell, a monocyte lineagecell type (macrophages, dendritic cells, Langerhans cells, mast cells),a fibroblast. In various embodiments, at least 5% of the cancer cellstransduced or transfected with the novel vector/vector system or a virusparticle disclosed herein are expressing 4-1BBL. The novel vector/vectorsystem or the virus particles of the present disclosure can be used inmethods for treating or ameliorating cancer, viral infections and/orimmune system disorders. Also, the polynucleotide of the presentdisclosure can be used in the treatment of cancer, viral infectionsand/or immune system disorders. Also, the above described composition ormedicament of the disclosure can be used in the treatment of cancers,viral infections and/or immune system disorders. In various embodiments,the novel vector/vector system or virus particles of the disclosure canbe considered as active agents. Also, in various embodiments thepolynucleotide of the disclosure, i.e., a polynucleotide comprising anucleic acid sequence encoding the novel vector/vector system of thedisclosure, and the cancer cell transduced or transfected with the novelvector/vector system or a virus particle disclosed herein, can beconsidered as active agents. This applies in particular in the contextof the medical treatments disclosed herein.

The present disclosure provides a method for treating or amelioratingcancer, a viral infectious disease (viral infection), or an immunesystem disorder comprising administering to a subject in need thereof atherapeutically effective amount of the novel vector/vector system or avirus particle of the disclosure. The present disclosure also provides amethod for treating or ameliorating cancer, a viral infectious disease(viral infection), or an immune system disorder comprising administeringto a subject in need thereof a therapeutically effective amount of apolynucleotide of the disclosure, i.e., a polynucleotide comprising anucleic acid sequence encoding the novel vector/vector system of thedisclosure. The present disclosure also provides a method for treatingor ameliorating cancer, a viral infectious disease (viral infection), oran immune system disorder comprising administering to a subject in needthereof a therapeutically effective amount of an immune cell or cancercell of the disclosure, i.e., an immune cell or cancer cell transducedor transfected with the novel vector/vector system or a virus particledisclosed herein. In various embodiments, the method of treating orameliorating cancer is performed as ex vivo therapy comprising obtainingcancer cells from a patient, transducing or transfecting the autologouscancer cells with a vector system or a virus particle disclosed herein,and administering the autologous cancer cells transduced or transfectedwith the novel vector/vector system or a virus particle disclosed hereinto the patient. The present disclosure also provides a method fortreating or ameliorating cancer, a viral infectious disease (viralinfection), or an immune system disorder comprising administering to asubject in need thereof a therapeutically effective amount of amedicament or composition of the disclosure described above.

As used herein, the term “therapeutically effective amount” refers to anamount of an active agent, composition or medicament disclosed herein,with which the harmful effects of a disease or disorder (e.g., cancer oran infectious disease) are, at a minimum, ameliorated.

In preferred embodiments, the cancer is any one of breast cancer,prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer,melanoma, malignant melanoma, ovarian cancer, brain cancer, primarybrain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer,bladder cancer, non-small cell lung cancer, head or neck carcinoma,breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lungcarcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma,bladder carcinoma, pancreatic carcinoma, stomach carcinoma, coloncarcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, chronic myelogenousleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, mesothelioma, osteogenicsarcoma, primary macro globulinemia, and retinoblastoma.

In other preferred embodiments, the cancer is any one of melanoma,cancer metastasis, adenocarcinoma, thyoma, lymphoma, sarcoma, lungcancer, colon cancer, Hodgkins lymphoma, uterine cancer, breast cancer,prostate cancer, ovarian cancer, cervical cancer, kidney cancer, andpancreatic cancer. In particularly preferred embodiments, the cancer isan urogenital cancer, preferably bladder cancer. In other particularlypreferred embodiments, the cancer is liver cancer. In still otherparticularly preferred embodiments, the cancer is skin cancer. The meansand methods provided by the present disclosure are also particularlyuseful in methods for treating or preventing cancer metastasis.

In various embodiments, the cancer comprises one or more tumors that areaccessible to allow for direct injection either into or around the tumorof an active agent (e.g., a vector system or virus particle),composition or medicament of the present disclosure. In particularlypreferred embodiments, the cancer comprises a solid tumor or is a solidtumor. In certain embodiments, the solid tumor is a carcinoma, a sarcomaor a lymphoma. In this regard, while lymphomas are generally consideredliquid tumors, accessible “solid” tumors may form in the lymph node andthus may be treated according to the methods and uses disclosed herein.

In various embodiments, the infectious disease is an infectious diseasecaused by a pathogenic bacterium. In other embodiments, the infectiousdisease is an infectious disease caused by a virus. In still otherembodiments, the infectious disease is an infectious disease caused by apathogenic parasite, protozoa or fungi.

Viral diseases that can be treated, protected against, and/or managedaccording to the present disclosure include, but are not limited to,those caused by hepatitis type A, hepatitis type B, hepatitis type C,influenza (e.g., influenza A or influenza B), varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papova virus, cytomegalovirus, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus,polio virus, small pox, Epstein Barr virus, human immunodeficiency virustype I (HIV-I), human immunodeficiency virus type II (HIV-II), Ebola,Zika, and agents of viral diseases such as viral meningitis,encephalitis, dengue or small pox.

Bacterial diseases caused by bacteria that can be treated, protectedagainst and/or managed in accordance with the present disclosureinclude, but are not limited to, Lyme disease, anthrax, tetanus,cholera, plague, diptheria, chlamydia, and pertussis.

Protozoan diseases caused by protozoa that can be treated, protectedagainst, and/or managed in accordance with the present disclosureinclude, but are not limited to, leishmania and malaria. Parasiticdiseases caused by parasites that can be treated, protected against,and/or managed in accordance with the present disclosure include, butare not limited to, chlamydia and rickettsia.

In the present disclosure, an immune system disorder is characterized bya downregulation of the immune system, in particular a downregulation ofthe immune response. The novel vector/vector system of the presentdisclosure enables control of such immune system disorders by convertingan inactive into an active immune microenvironment and thereby treatingan immune system disorder.

Likewise, in the present disclosure, cancer may be characterized by adownregulation of the immune system, in particular a downregulation ofthe immune response. Thus, the novel vector/vector system of the presentdisclosure enables control or treatment of cancer by converting aninactive into an active tumor microenvironment and thereby treating orcontrolling cancer.

Also, in the present disclosure, an infectious disease may becharacterized by a downregulation of the immune system, in particular adownregulation of the immune response. Thus, the novel vector/vectorsystem of the present disclosure enables control or treatment ofinfectious diseases by converting an inactive into an active immunemicroenvironment and thereby treating or controlling infectiousdiseases.

In various embodiments of the present disclosure, the vector system ispresent in a concentration of not more than 1×10¹¹ ivp, preferably notmore than 1×10¹⁰ ivp, more preferably not more than 1×10⁹ ivp, even morepreferably not more than 1×10⁷ or 1×10⁶ ivp per dose unit. This appliesin particular to, but is not limited to, the medical treatmentsdisclosed herein.

Furthermore, in various embodiments of the present disclosure the virusparticle is present in a concentration of not more than 1×10¹¹ ivp,preferably not more than 1×10¹⁰ ivp, more preferably not more than 1×10⁹ivp, even more preferably not more than 1×10⁷ or 1×10⁶ ivp per doseunit. This applies in particular to, but is not limited to, the medicaltreatments disclosed herein.

In various embodiments, 5×10⁶ ivp (infectious viral particles) of thenovel vector/vector system or virus particle of the present disclosureis administered to a patient in need thereof. In various otherembodiments, 5×10⁷ ivp of the novel vector/vector system or virusparticle of the present disclosure is administered to a patient in needthereof. In various other embodiments, 5×10⁸ ivp of the novelvector/vector system or virus particle of the present disclosure isadministered to a patient in need thereof.

In the present disclosure, the terms “medicament” or “pharmaceuticalcomposition” may be used interchangeably. The medicament orpharmaceutical composition may be in any form, such as solid, semisolidor liquid form, suitable for administration. A formulation can be anyone of, but not limited to, a solution, emulsion or suspension. Meansand methods for formulating the present pharmaceutical preparations areknown to persons skilled in the art, and may be manufactured in amanner, which is in itself known. The medicament (or pharmaceuticalcomposition) may be administered in combination with a pharmaceuticallyacceptable carrier, excipient or diluent. Pharmaceutically acceptablecarriers are well known in the art and include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, amino acids, sterileisotonic aqueous buffer, and combinations thereof.

The active agents (e.g., the novel vector/vector system or viralparticles), compositions and medicaments of the present disclosure maybe administered parenterally. Solutions or suspensions of these activecompounds can be prepared in water suitably mixed with a surfactant,such as hydroxypropylcellulose. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof in oils.Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solution,and glycols such as, propylene glycol or polyethylene glycol, arepreferred liquid carriers, particularly for injectable solutions. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. Formulations forparenteral and nonparenteral drug delivery are known in the art and areset forth in Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing (1995), which is hereby incorporated by reference in itsentirety.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Subjects to be treated in accordance with the present disclosure aresubjects that are at risk of developing, or have developed, cancer, aninfectious disease, or an immune system disorder. Such subjects includehuman and non-human animals, preferably mammals or avian species.Exemplary mammalian subjects include, without limitation, humans,non-human primates, dogs, cats, rodents, cattle, horses, sheep, andpigs. Exemplary avian subjects include, without limitation, chicken,quail, turkey, duck or goose.

An effective amount of a therapeutic or preventive active agent,composition or medicament of the disclosure is determined based on theintended goal, for example stimulation of an immune response against atumor or an infectious disease. Those of skill in the art are well awareof how to apply gene delivery in vivo and ex vivo. For viral vectors,one generally will prepare a viral vector stock. Depending on the kindof virus and the titer attainable, one will deliver at least about, atmost about, or about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹ or 1×10¹² infectious viral particles, or any value or range therebetween, to a subject. In other aspects, the viral vector(s) accordingto the present disclosure may be administered in a single administrationor multiple administrations. The viral vector(s) may be administered atdosage of 1×10⁵ infectious viral particles (ivp), 5×105 ivp, at least1×10⁶ivp, 5×10⁶ or about 5×106 ivp, 1×10⁷, at least 1×107 ivp, 1×10⁸ orabout 1×108 ivp, at least 1×108 ivp, about or at least 5×10⁸ ivp, 1×10⁹or at least 1×10⁹ ivp, 5×10⁹ or at least 5×10⁹ ivp, 1×10¹⁰ ivp or atleast 1×1010 ivp, 5×10¹⁰ or at least 5×10¹⁰ ivp, 1×10¹¹ or at least1×10¹¹, 1×10¹² or at least 1×10¹², 1×10¹³ or at least 1×10¹³ ivp. Forexample, the viral vector(s) may be administered at a dosage of betweenabout 10⁷-10¹³ ivp, between about 10⁸-10¹³ ivp, between about 10⁸-10¹²ivp, or between about 10⁹-10¹² ivp.

A therapeutic effect may be achieved with only one administration of anactive agent (e.g., a vector system or virus particle), composition ormedicament of the present disclosure. On the other hand, the treatmentmay contain several administrations.

The effective dose of vectors depends on at least the subject in need ofthe treatment, type of the disease and stage of the disease. The dosemay vary for example from about 1×10⁸ ivp (infectious viral particles)to about 1×10¹⁴ ivp, specifically from about 1×10⁹ ivp to about 1×10¹³ivp, and more specifically from about 5×10⁹ ivp to about 1×10¹² ivp.

Administration of the active agent (e.g., a vector system or virusparticle), composition or medicament of the present disclosure can beconducted through any suitable method known to a person skilled in theart. In one embodiment of the disclosure, the administration isconducted through an intratumoral, intra-arterial, intravenous,intrapleural, intravesicular, intracavitary or peritoneal injection, oran oral administration. In another embodiment of the disclosure, theadministration is conducted intrasmuscularly, intradermally,subcutaneously, parenterally, intranasally, intratracheally,percutaneously, intraspinally, ocularly, or intracranially. It is alsopossible to combine different routes of administration. In a preferredembodiment, the administration is conducted through an intratumoraladministration, i.e., administration of the active agent (e.g., a vectorsystem or virus particle), composition or medicament of the presentdisclosure into the tumor.

The active agent (e.g., a vector system or virus particle), compositionor medicament of the present disclosure may also be used together(simultaneously, sequentially, or concomitantly) with other therapeuticagents or therapeutic methods or a combination of treatments. Forexample, the therapeutic methods or uses of the disclosure may furthercomprise radiotherapy, chemotherapy, administration of other drugs, e.g.antibodies addressing tumor growth mechanisms, immune cell checkpointtargets, cancer vaccines, or any clinical operations.

As described herein, methods and uses are provided for immunostimulation(i.e., inducing, controlling and/or activating and/or stimulating immuneresponse mechanisms in the context of the treatment of cancer, aninfectious disease or an immune system disorder, in particular forattracting or recruiting cells induced, activated and/or stimulated bythe immunostimulation to a site of interest (e.g., to a tumor, or amucosal site of infection). Cells of the immune system that are involvedin an immune response are referred to, generally, as immune cells andinclude lymphocytes and non-lymphoid cells such as accessory cells.Lymphocytes are cells that specifically recognize and respond to foreignantigens. Major classes of lymphocytes include B lymphocytes (B cells),T lymphocytes (T cells), and natural killer (NK) cells, which are largegranular lymphocytes. B cells are capable of producing antibodies. Tlymphocytes are further subdivided and include helper T cells (CD4+ Tcells) and cytolytic or cytotoxic T cells (CD8+ T cells). Helper cellssecrete cytokines that promote proliferation and differentiation of theT cells and other cells, including B cells and macrophages, and recruitand activate inflammatory leukocytes. Another subgroup of T cells,called regulatory T cells or suppressor T cells, actively suppressactivation of the immune system and prevent pathologicalself-reactivity, that is, autoimmune disease.

The immunostimulation methods described herein is considered to induce acell-mediated immune response involving various types of T cells. In acell mediated response, the various types of T lymphocytes act toeliminate an antigen by a number of mechanisms. For example, helper Tcells that are capable of recognizing specific antigens may respond byreleasing soluble mediators such as cytokines to recruit additionalcells of the immune system to participate in an immune response. Also,cytotoxic T cells are capable of specifically recognizing an antigen andmay respond by binding to and destroying or damaging an antigen-bearingcell or particle.

An immune response in a host or subject may be determined by any numberof well-known immunological methods with which those having ordinaryskill in the art will be familiar. As described herein, methods andtechniques for determining the presence and level of an immune responseinclude, for example, fluorescence resonance energy transfer,fluorescence polarization, time-resolved fluorescence resonance energytransfer, scintillation proximity assays, reporter gene assays,fluorescence quenched enzyme substrate, chromogenic enzyme substrate andelectrochemiluminescence, immunoassays, (such as enzyme-linkedimmunosorbant assays (ELISA), radioimmunoassay, immunoblotting,immunohistochemistry, and the like), surface plasmon resonance,cell-based assays such as those that use reporter genes, and functionalassays (e.g., assays that measure immune function andimmunoresponsiveness).

Such assays include, but need not be limited to, in vivo or in vitrodetermination of the presence and level of soluble antibodies, solublemediators such as cytokines (e.g., IFN-γ, IL-2, IL-4, IL-10, IL-12,IL-6, IL-23, TNF-α, and TGF-8), lymphokines, chemokines, hormones,growth factors, and the like, as well as other soluble small peptide,carbohydrate, nucleotide and/or lipid mediators. Levels of cytokines maybe determined according to methods described and practiced in the art,including, for example, ELISA, ELISPOT, intracellular cytokine staining,and flow cytometry and combinations thereof (e.g., intracellularcytokine staining and flow cytometry).

Immunoassays also include determining cellular activation state changesby analyzing altered functional or structural properties of cells of theimmune system, for example, cell proliferation, altered motility,induction of specialized activities such as specific gene expression orcytolytic behavior; cell maturation, such as maturation of dendriticcells in response to a stimulus; alteration in relationship between aTh1 response and a Th2 response; cellular differentiation by cells ofthe immune system, including altered surface antigen expression profilesor the onset of apoptosis (programmed cell death). Other methods arealso available for measuring cell surface markers to identify variouspopulations of immune cells, such as, but not limited to,antigen-specific CD4+ and/or CD8+ T cells, effector memory T cells(Tem), central memory T cells (Tcm) and/or tissue-resident memory Tcells (Trm). Procedures for performing these and similar assays aredescribed in the literature. Cytotoxicity assays for determining CTLactivity (or CD8+ T cell activity) may be performed using any one ofseveral techniques and methods routinely practiced in the art.

In particular embodiments, a 2-50 fold increase in locally infiltratingantigen-specific T cells is observed following the methods and usesdisclosed herein. In certain embodiments, a 2-40 fold increase, a 2-30fold increase, a 2-20 fold increase, a 2-10 fold increase, 3-8 foldincrease, a 4-7 fold increase, or a 5-6 fold increase in locallyinfiltrating (e.g., tumor-infiltrating) antigen-specific T cells isobserved. Generally, the increase in locally infiltratingantigen-specific T cells is as compared to the number of locallyinfiltrating antigen-specific T cells present in the absence ofadministration or as compared to an appropriate control administration.The methods and uses disclosed herein are considered to provide anincrease in a statistically, biologically, and/or clinically significantmanner of the locally infiltrating antigen-specific T cells as comparedto an appropriate control in the absence of administering the activeagent, composition or medicament of the disclosure.

A biological sample may be obtained from the subject for determining thepresence and level of an immune response in the subject who has receiveda treatment with an active agent (e.g., a vector system or virusparticle), composition or medicament of the disclosure according to themethods disclosed herein. A “biological sample” as used herein may be ablood sample (from which serum or plasma may be prepared), apheresissample, biopsy specimen, tumor biopsy specimen, body fluids (e.g., lunglavage, ascites, mucosal washings, synovial fluid), bone marrow, lymphnodes, tissue explant, organ culture, or any other tissue or cellpreparation from the subject or a biological source.

With respect to all immunoassays and methods described herein fordetermining an immune response, a person skilled in the art will alsoreadily appreciate and understand which controls are appropriatelyincluded when practicing these methods. Concentrations of reactioncomponents, buffers, temperature, and time period sufficient to permitinteraction of the reaction components can be determined and/or adjustedaccording to methods with which a person skilled in the art is familiar.

Another aspect of the present disclosure provides a method of increasingT-cells in the tumor microenvironment comprising administering to asubject having a tumor an active agent (e.g., a vector system or virusparticle), composition or medicament of the disclosure according to themethods disclosed herein, thereby inducing an immune response againstthe tumor.

As understood by a person skilled in the medical art, the terms “treat”and “treatment” refer to medical management of a disease, disorder, orcondition of a subject (i.e., patient). In general, an appropriate doseand treatment regimen provide the active agent (e.g., a vector system orvirus particle), composition or medicament of the disclosure in anamount sufficient to provide therapeutic and/or prophylactic benefit.Therapeutic and/or prophylactic benefit includes, for example, animproved clinical outcome, both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow orretard (lessen) an undesired physiological change or disorder, or toprevent or slow or retard (lessen) the expansion or severity of suchdisease or disorder. Beneficial or desired clinical results fromtreating a subject include, but are not limited to, abatement,lessening, or alleviation of symptoms that result from or are associatedwith the disease or disorder to be treated; decreased occurrence ofsymptoms; improved quality of life; longer disease-free status (i.e.,decreasing the likelihood or the propensity that a subject will presentsymptoms on the basis of which a diagnosis of a disease is made);diminishment of extent of disease; stabilized (i.e., not worsening)state of disease; delay or slowing of disease progression; ameliorationor palliation of the disease state; and remission (whether partial ortotal), whether detectable or undetectable; and/or overall survival.“Treatment” can also mean prolonging survival when compared to expectedsurvival if a subject were not receiving treatment. Subjects in need oftreatment include those who already have the disease or disorder as wellas subjects prone to have or at risk of developing the disease ordisorder).

Nucleic acid molecules, including vector systems according to thepresent disclosure, may be delivered into a cell according to any one ofseveral methods described in the art. Such delivery methods known topersons having skill in the art, include, but are not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as biodegradable polymers; hydrogels;cyclodextrins; poly(lactic-co-glycolic)acid (PLGA) and PLCAmicrospheres; biodegradable nanocapsules; and bioadhesive microspheres,or by proteinaceous vectors.

The present disclosure provides a combination of proteins comprising4-1BB ligand (4-1BBL), IL-2 and single chain IL-12 (scIL-12), whereinthe amount of 4-1BBL is higher than the amount of scIL-12 and IL-2.

In various embodiments, the 4-1BB ligand comprises an amino acidsequence having at least 70% homology or identity to the amino acidsequence of SEQ ID NO: 2 (FIG. 13), wherein the 4-1BB ligand is capableof specifically binding T cells, preferably activated CD4+ T helpercells and CD8+ T cells. Preferably, the 4-1BB ligand comprises an aminoacid sequence having at least 80% homology or identity to the amino acidsequence of SEQ ID NO: 2 (FIG. 13), wherein the 4-1BB ligand is capableof specifically binding T cells, preferably CD8+ T cells. Morepreferably, the 4-1BB ligand comprises an amino acid sequence having atleast 90% homology or identity to the amino acid sequence of SEQ ID NO:2 (FIG. 13), wherein the 4-1BB ligand is capable of specifically bindingT cells, preferably activated CD4+ T helper cells and CD8+ T cells. Evenmore preferably, the 4-1BB ligand comprises an amino acid sequencehaving at least 95% homology or identity to the amino acid sequence ofSEQ ID NO: 2 (FIG. 13), wherein the 4-1BB ligand is capable ofspecifically binding T cells, preferably activated CD4+ T helper cellsand CD8+ T cells. In certain embodiments, variants of 4-1BBL asdescribed above exhibit the same binding specificity for T cells,preferably for activated CD4+ T helper cells and CD8+ T cells, as thenative 4-1BBL having the amino acid sequence of SEQ ID NO: 2 (FIG. 13).

In various embodiments of the disclosure, the IL-2 protein shows atleast 70% homology or sequence identity to the amino acid sequence ofSEQ ID NO: 4 (FIG. 14), wherein the IL-2 protein has immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity.Preferably, the IL-2 protein shows at least 80% homology or sequenceidentity to the amino acid sequence of SEQ ID NO: 4 (FIG. 14), whereinthe IL-2 protein has immune stimulating activity, preferably T helpercell and CD8+ T cell stimulating activity. More preferably, the IL-2protein shows at least 90% homology or sequence identity to the aminoacid sequence of SEQ ID NO: 4 (FIG. 14), wherein the IL-2 protein hasimmune stimulating activity, preferably T helper cell and CD8+ T cellstimulating activity. Even more preferably, the IL-2 protein shows atleast 95% homology or sequence identity to the amino acid sequence ofSEQ ID NO: 4 (FIG. 14), wherein the IL-2 protein has immune stimulatingactivity, preferably T helper cell and CD8+ T cell stimulating activity.In certain embodiments, variants of IL-2 as described above exhibit thesame immune stimulating activity, preferably T helper cell and CD8+ Tcell stimulating activity, as the native IL-2 having the amino acidsequence of SEQ ID NO: 4 (FIG. 14).

In various embodiments of the disclosure, the scIL-12 protein comprisesan amino acid sequence having at least 70% homology or sequence identityto the amino acid sequence of SEQ ID NO: 6 (FIG. 15), wherein thescIL-12 protein has immune stimulating activity, preferably monocyte, Thelper cell and CD8+ T cell stimulating activity. Preferably, thescIL-12 protein comprises an amino acid sequence having at least 80%homology or sequence identity to the amino acid sequence of SEQ ID NO: 6(FIG. 15), wherein the scIL-12 protein has immune stimulating activity,preferably monocyte, T helper cell and CD8+ T cell stimulating activity.More preferably, the scIL-12 protein comprises an amino acid sequencehaving at least 90% homology or sequence identity to the amino acidsequence of SEQ ID NO: 6 (FIG. 15), wherein the scIL-12 protein hasimmune stimulating activity, preferably monocyte, T helper cell and CD8+T cell stimulating activity. Even more preferably, the scIL-12 proteincomprises an amino acid sequence having at least 95% homology orsequence identity to the amino acid sequence of SEQ ID NO: 6 (FIG. 15),wherein the scIL-12 protein has immune stimulating activity, preferablymonocyte, T helper cell and CD8+ T cell stimulating activity. In certainembodiments, variants of scIL-2 as described above exhibit the sameimmune stimulating activity, preferably monocyte, T helper cell and CD8+T cell stimulating activity, as the native scIL-2 having the amino acidsequence of SEQ ID NO: 6 (FIG. 15).

As disclosed herein, a protein is considered a scIL-12 protein if itcomprises an amino acid sequence comprising the two subunits p35 and p40of the native IL-12 protein as a fusion protein. The sequences of SEQ IDNOs: 8 and 10 show the amino acid sequence of the 40 kDa and 35 kDasubunits of human IL-12. In certain embodiments, variants of scIL-12 asdescribed above exhibit the same immune stimulating activity as thenative scIL-12 encoded by the amino acid sequences of SEQ ID NOs: 8 and10. Preferably, the linker of the scIL-12 of the present disclosure is apeptide or polypeptide linker. The present disclosure encompassesvariants of the scIL-12 as described herein, in which in particular thelinker sequence shown in boldface in FIG. 15 (SEQ ID NOs: 5 and 6) ismodified, specifically with respect to the length of the linkersequence. The length of the amino acid sequence of the linker can beselected empirically or with guidance from structural information or byusing a combination of the two approaches. Those skilled in the art willrecognize that there are many such sequences that vary in length orcomposition that can serve as linkers with the primary considerationbeing that they be neither excessively long nor short.

The novel vector/vector system or virus particles provided by thepresent disclosure can be packaged as kits. Kits can optionally includeone or more components such as instructions for use and administration,devices (e.g., for administering the composition or compositions to asubject), and additional reagents, and components, such as tubes,containers, e.g. vials, and syringes for practice of the methods anduses. Kits comprising a polynucleotide comprising a nucleic acidsequence encoding a vector system of the disclosure are alsocontemplated herein. Kits comprising a cancer cell transduced ortransfected with a vector system or virus particle of the disclosure arealso contemplated herein. Kits comprising an active agent, acomposition, or a medicament of the disclosure are also contemplatedherein.

Kits comprising the novel viral vector/vector system of the disclosureand optionally a polynucleotide sequence encoding a maturation factorare also contemplated herein.

In light of the present disclosure, herein encompassed are, withoutbeing limited thereto, the following items, which are to be consideredin the context of the aspects and embodiments described elsewhereherein:

1. Vector comprising nucleic acid sequences of genes encoding 4-1BBligand (4-1BBL), single chain IL-12 (scIL-12) and IL-2, wherein the saidgenes are organized in 5′ to 3′ orientation in a sequential order 1, 2,3, with the proviso that the gene encoding scIL-12 is not at position 1.2. The vector of item 1, wherein the vector is any one of an adenoviralvector, an adeno-associated virus vector, a lentiviral vector, aretroviral vector, a herpes simplex virus vector, a pox virus vector, aRNA vector, a plasmid vector, a nanoparticle vector, and naked DNA.3. The vector of item 2, wherein the RNA vector comprises insertedmodified ribonucleotides.4. The vector of any one of items 1-3, wherein the nucleic acid sequenceof the gene encoding 4-1BBL is human cDNA, the nucleic acid sequence ofthe gene encoding scIL-12 is human cDNA, and/or the nucleic acidsequence of the gene encoding IL-2 is human cDNA.5. The vector of any one of items 1-4, wherein the nucleic acid sequenceof the gene encoding 4-1BBL shows at least 70% homology or sequenceidentity to the nucleic acid sequence of SEQ ID NO: 1 (FIG. 13), whereinthe variant nucleic acid sequence encodes a 4-1BBL protein capable ofspecifically binding T cells, preferably activated T cells.6. The vector of any one of items 1-4, wherein the nucleic acid sequenceof the gene encoding IL-2 shows at least 70% homology or sequenceidentity to the nucleic acid sequence of SEQ ID NO: 3 (FIG. 14), whereinthe variant nucleic acid sequence encodes a IL-2 protein having immunestimulating activity.7. The vector of any one of items 1-4, wherein the nucleic acid sequenceof the gene encoding scIL-12 shows at least 70% homology or sequenceidentity to the nucleic acid sequence of SEQ ID NO: 5 (FIG. 15), whereinthe variant nucleic acid sequence encodes an scIL-12 protein havingimmune stimulating activity.8. The vector of any one of items 1-7, wherein the nucleic acidsequences of the genes encoding scIL-12 and IL-2 are located downstreamof the nucleic acid sequence of the gene encoding 4-1BBL.9. The vector of item 8, wherein the nucleic acid sequence of the geneencoding IL-2 is located downstream of the nucleic acid sequence of thegene encoding 4-1BBL, and the nucleic acid sequence of the gene encodingscIL-12 is located downstream of the nucleic acid sequence encodingIL-2.10. The vector of item 8 or 9, wherein a promoter is located upstream ofthe nucleic acid sequence of the gene encoding 4-1BBL, but not upstreamof the nucleic acid sequences of the genes encoding scIL-12 and/or IL-2.11. The vector of any one of items 8-10, wherein the nucleic acidsequences of the genes encoding 4-1BBL, scIL-12 and IL-2 are linked byinternal ribosomal entry sites (IRES).12. Virus particle comprising the vector of any one of items 1-11.13. A polynucleotide comprising a nucleic acid sequence encoding thevector of any one of items 1-11.14. A cancer cell or an immune cell, transduced or transfected with thevector of any one of items 1-11 or the virus particle of item 12.15. A composition comprising the vector of any one of items 1-11, thevirus particle of item 12, the polynucleotide of item 13, or the cancercell or immune cell of item 14.16. A medicament comprising the vector of any one of items 1-11, thevirus particle of item 12, the polynucleotide of item 13, or the cancercell or immune cell of item 14.17. The vector of any one of items 1-11, the virus particle of item 12,the polynucleotide of item 13, the cancer cell or immune cell of item14, the composition of item 15, or the medicament of item 16 for use ina method of treating cancer, a viral infection and/or an immune systemdisorder.18. The vector for use according to item 17, the virus particle for useaccording to item 17, the polynucleotide for use according to item 17,the cancer cell or immune cell for use according to item 17, thecomposition for use according to item 17, or the medicament for useaccording to item 17, wherein the cancer is any one of breast cancer,prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer,melanoma, malignant melanoma, ovarian cancer, brain cancer, primarybrain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer,bladder cancer, non-small cell lung cancer, head or neck carcinoma,breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lungcarcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma,bladder carcinoma, pancreatic carcinoma, stomach carcinoma, coloncarcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, chronic myelogenousleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, mesothelioma, osteogenicsarcoma, primary macro globulinemia, and retinoblastoma.19. The vector of any one of items 1-11, the virus particle of item 12,the composition of item 13, or the medicament of item 14 for use in amethod of preventing or treating cancer metastasis.20. The vector for use according to any one of items 17-19,characterized in that the vector system is present in a concentration ofnot more than 1×10¹¹ ivp (infectious viral particles), preferably notmore than 1×10¹⁰ ivp, more preferably not more than 1×10⁹ ivp, even morepreferably not more than 1×10⁷ ivp or 1×10⁶ ivp per dose unit.21. The virus particle for use according to any one of items 17-19,characterized in that the virus particle is present in a concentrationof not more than 1×10¹¹ ivp, preferably not more than 1×10¹⁰ ivp, morepreferably not more than 1×10⁹ ivp, even more preferably not more than1×10⁷ ivp or 1×10⁶ ivp per dose unit.22. A combination of proteins comprising 4-1BB ligand (4-1BBL), IL-2 andsingle chain IL-12 (scIL-12), wherein the amount of 4-1BBL is higherthan the amount of scIL-12 and IL-2

It is to be acknowledged that the present disclosure is not limited tothe particular methodology, protocols, cell lines, genera, and reagentsdescribed herein, as such may vary. It is also to be acknowledged thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting the scope of thepresent disclosure.

The following examples are offered by way of illustration and not by wayof limitation.

Other embodiments and uses will be apparent to one skilled in the art inlight of the present disclosures. The following examples are providedmerely as illustrative of various embodiments and shall not be construedto limit the present disclosure in any way.

EXAMPLES Example 1: Transgene Expression of IL-12, IL-2 and 4-1BBL andIFN-γ Response of Murine and Human Im01

Im01 is an adenoviral vector comprising an expression constructcomprising the human genes for single chain IL-12, 4-1BBL, and IL-2 inthe order as shown in the following scheme:

-CMV>-|scIL-12|-(IRES)-|4-1BBL|-(IRES)-|IL-2|-

The construction of vector Im01 is described in WO 2004/035799 with theabove scheme depicted in FIG. 1 of WO 2004/035799. The vector name in WO2004/036799 is “Ad-3”. In the present disclosure, the internal vectorcode for the earlier vector is Im01.

Human A549 cells and murine Hepa1-6 cells were transduced one hour withIm01 carrying the three human or mouse genes, respectively, at theindicated multiplicity of infection (MOI, numbers given in [brackets]).Human peripheral blood mononuclear cells (PBMCs) or mouse lymphocyteswere added 4 hours after transduction and supernatants were collectedfor cytokine assays after 34 hours of co-culture. Cytokine levels weredetected by ELISA (eBioscience). Tumor cells were detached and assayedby flow-cytometry for 4-1BBL expression. As a result, even though themurine and human vector architecture is identical, the expression levelof the transgenes clearly varies between murine and human species inthat the human Im01 shows up to 15-fold higher IL-12 expression than themurine Im01. In the clinical setting such increased IL-12 levels carrythe risk for toxicity and for limited therapeutic applicability.

Example 2: Vector Design and Study Overview Im02

A vector according to the present disclosure has been designed andproduced for an ex vivo therapy simulation study. The vector is based onan adenoviral vector and has been named Im02 (internal vector code“Im02”). Im02 comprises an expression construct comprising the humangenes for 4-1BBL, IL-2 and single chain IL-12 (scIL-12) in the order asshown in the following scheme:

-CMV>-|4-1BBL|-(IRES)-|IL-2|-(IRES)-|scIL-12|-

FIG. 2 shows a schematic gene map of the shuttle plasmid pE1.1 Im02. Theexpression cassette for Im02 is illustrated as a precursor transferplasmid based on plasmid pE1.1, suitable for sub-cloning into a plasmidcarrying, e.g., adenoviral vector DNA. More specifically, the expressioncassette contained in the adenoviral vector Im02 is the expressioncassette as shown in FIG. 18 comprising the nucleotide sequence of SEQID NO: 12. The vector of the present disclosure has been identified toinduce immune defense mechanisms against tumor cells in bladder cancerby multivalent modification of the intratumoral immune microenvironment.

Patient Cohort Overview

Ex vivo cultures of human tumor biopsy samples were chosen as studymodel for the novel vector system. Samples were provided by the Clinicof Urology, Asklepios hospital, Hamburg-Barmbek. In total 244 tumorsamples and 270 normal bladder tissue control biopsies from 43 patientshave been analyzed. From eight patients samples were collected duringtransurethral resection (TUR), the major part of the study-includedsamples was from cystectomy. In this study, tumor and bladder tissueswere analyzed across a wide range of disease stages, varying from earlyto late stage. Samples from 16 females and 27 males have been analyzed.Pretreatments in this patient cohort were serial TUR, chemotherapy, oranti-androgen therapy, due to concomitant diagnosis of prostatecarcinoma.

Example 3: Transgene Expression of 4-1BBL, IL-2 and scIL-12, and IFN-γResponse of Im02 and the Earlier Vector Im01

Transgene expression of 4-1BBL, IL-2 and IL-12, and IFN-γ response ofIm02 (see Example 2) and the earlier vector Im01 (see Example 1) werecompared.

Human A549-cells were transduced one hour with Im01 or Im02 at the MOI(multiplicity of infection, i.e., infectious viral particles per targetcell) indicated in FIG. 3 (MOI numbers in brackets). Human peripheralblood mononuclear cells (PBMCs) were added 4 hours after transductionand supernatants were collected for cytokine assays after 34 hours ofco-culture. Cytokine levels were detected by ELISA. Tumor cells weredetached and cells positive for 4-1BBL were detected by flow cytometry.Indicated data points are the mean of four individual donors each withfour replicates.

As shown in FIG. 3, the arrangement of the genes for 4-1BBL, IL-2 andscIL-12 in Im02 provides for an increased expression of 4-1BBL ascompared to the arrangement of the same genes in Im01, concurrent with adecrease of IL-12, leading to an increase in IFN-γ response. Inparticular, the arrangement of the genes for 4-1BBL, IL-2 and scIL-12 inIm02 leads to a 1.7-fold increase of 4-1BBL expression, and a 1.5-foldincrease of IL-2 expression in mean, as compared to the arrangement ofgenes in the earlier vector Im01. This is combined with a 2.6-folddecrease of the IL-12 expression level. With the arrangement of thegenes in Im02, the molar ratio of IL-2/IL-12 was increased from 2.5% to9.1% on average. Furthermore, higher IL-2 and 4-1BBL expression leads toa 1.4-fold higher IFN-γ induction detected at all dose ranges, MOI 2.5,5, 10, and 50.

Im02 provides for an IFN-γ response, which is superior over that ofIm01. As a result, Im02 shows an improved effect in immunostimulation ascompared to the earlier vector Im01.

Example 4: Comparison of Single Vectors and Im02 and Im01 in aTissue-Based Model

Single-dose treatments using adenoviral vectors expressing scIL-12, IL-2or 4-1BBL alone were examined as well as Im02 and Im01 (see Example 1for further details of the latter one). To study the Im02 effects on thetumor microenvironment, a therapy simulation model based onundissociated tumor tissue samples was established. Tissue samplesderived from transurethral resection or bladder cystectomy were used.

Bladder tumor (“T”) and normal bladder (“B”) tissues were transducedwith 10⁸ ivp (infectious virus particles) of Im02 or Im01. Viability oftumor and bladder tissue samples was monitored in culture mediumsupernatants using an enzymatic LDH-release assay. Expression oftransgenes and IFN-γ response was measured by ELISA in culturesupernatant at day 6 after transduction. The results are shown in FIG.4. Even at high expression levels of scIL-12 and IL-2 alone the IFN-γresponse is close to background. This result suggests the cooperativeaction of the three genes in the tissue context of bladder and tumor.Furthermore, Im02 leads to an increase of IL-2 and IFN-γ response ascompared to Im01. FIG. 4 shows an improved effect for Im02 inimmunostimulation in the tumor microenvironment as compared to theearlier vector Im01.

Example 5: Comparison of Im02 and Im01 at Different Dose Levels

Im02 and Im01 (see Examples 2 and 3 for further details of thesevectors) were compared at escalating doses for transgene expression andIFN-γ response in the tumor microenvironment. Tumor samples were derivedfrom individual patients, and a matched pair of bladder tumor (“T”) andnormal bladder (“B”) tissues was examined for the dose-levels 10⁷ and10⁸ ivp (infectious virus particles). Expression was measured at day 6by ELISA. The results are shown in FIG. 5. At different dose levels,Im02 provides for an IFN-γ response, which is superior over that ofIm01. As a result, at different dose levels Im02 shows an improvedeffect in immunostimulation in the tumor microenvironment as compared tothe earlier vector Im01.

Example 6: Gene Expression Profiling for Im02 and Im01

A transcriptome analysis has been performed to show the therapeutic geneexpression profile for Im02. In particular, in order to obtain acomprehensive profile of leukocyte activation by Im02 and Im01, tumorcells of a co-culture experiment with tumor cells and PBMCs weretransduced with Im02, Im01, and Ad0 (empty vector), and a mRNA geneactivity analysis (Illumina Chip HT12 whole genome expression analysis)of the leukocytes was performed. To this end, peripheral leukocytes wereadded to the co-culture after transduction. Leukocytes on non-transducedtumor cells were used as control. After 4, 24, 32, 48, 72 and 96 hours,leukocytes were collected and the RNA was isolated and purified. Afterquality check, the RNA is reversely transcribed into complimentary DNA(cDNA), labeled according to the protocol of the bead chip and wasloaded onto the bead chip HT12 and a scan was performed (Life & Brain,department of human genetics at the University of Bonn). The obtaineddata were transferred to the GenomeStudio Software (Illumina).Subsequently, the data were evaluated using the IPA® Software(Ingenuity). A core analysis was performed showing the differentiallyregulated processes. In order to gain an overview of the regulatedprocesses, and to illustrate their significance and the involved numberof molecules, an IPA® process analysis was performed. See Tables 1 and2:

TABLE 1 Im02, the five most heavily regulated processes at 24 h #Mole-Name p-value cules Molecular and Cellular Functions Cell-to-CellSignaling and Interaction 1.28E−22 − 4.91E−03 103 Cellular Function andMaintenance 4.34E−22 − 4.52E−03  90 Cell Death and Survival 3.48E−17 −3.98E−03  69 Cellular Development 1.46E−16 − 5.71E−03 101 CellularGrowth and Proliferation 1.46E−16 − 5.71E−03  89 Physiological SystemDevelopment and Function Hematological System Development 1.28E−22 −5.71E−03 162 and Function Immune Cell Trafficking 1.28E−22 − 4.96E−03108 Tissue Morphology 9.81E−17 − 5.59E−03  86 Cell-mediated ImmuneResponse 6.66E−10 − 3.84E−03  61 Tissue Development 1.53E−08 − 4.91E−03 54

TABLE 2 Im01, the five most heavily regulated processes at 24 h #Mole-Name p-value cules Molecular and Cellular Functions Cell-to-CellSignalling and Interaction 8.05E−17 − 7.51E−03 61 Cell Death andSurvival 1.27E−11 − 7.77E−03 42 Cellular Function and Maintenance8.69E−11 − 7.16E−03 46 Cellular Growth and Proliferation 1.39E−09 −7.16E−03 47 Cellular Compromise 3.44E−08 − 3.39E−03 25 PhysiologicalSystem Development and Function Hematological System Development8.05E−17 − 7.51E−03 83 and Function Immune Cell Trafficking 8.05E−17 −7.51E−03 64 Tissue Development 4.10E−07 − 6.95E−03 31 Hematopoiesis1.04E−06 − 6.95E−03 35 Tissue Morphology 2.17E−06 − 6.95E−03 40

As a result, Im02 provides for an immunostimulation, which ischaracterized by a “cell-mediated immune response” involving 61regulated genes (see Table 1, lower part) among the five most heavilyregulated processes of physiological development and function (by virtueof significance and number of molecules involved). For Im01 (see Table2, lower part), this function (i.e., “cell-mediated immune response”)does not appear among the five most heavily regulated processes ofphysiological development and function because here only a total of 28molecules are differentially regulated. The other systems mentioned inthe table (like the hematological system, immune cell trafficking,tissue morphology, and general tissue development), suggest extensivechanges caused by multivalent immune therapeutics. What becomes clearfrom the transcriptome analysis is that the therapeutic gene expressionprofile for Im02 is unique and superior, in particular superior overthat of the earlier vector Im01. The regulated processes have beenallocated to specifically regulated (cell) functions using the IPA®software (Ingenuity Pathway Analysis, Qiagen). This permits moreaccurate information as regards the activation/inactivation ofbiological processes.

As a result (data not shown), the five most heavily induced functionsare lymphoycyte activation, activation of mononuclear leukocytes,cytotoxicity of leukocytes, differentiation of mononuclear leukocytes,and activation of T lymphocytes (T cells).

Example 7: Gene Expression Analysis for Activation of Major Immune CellTypes

The activation of major immune cell types over a time course of 96 hourshas been analyzed using the CELLMIX software, which allows analyzinggene expression data for the presence and state of activation of allmajor immune cell types. The heat plot in FIG. 6 illustrates theactivation of all major blood immune cell subtypes except for B-cells(peripheral blood mononuclear cells, PBMCs) over a time course of 4 days(96 hours) in co-culture with human bladder RT-4 carcinoma cells. Asshown in FIG. 6, the activation of T helper cells and cytotoxic T cellsby Im02 is superior over the activation of the same immune cells byIm01.

Example 8: Ex Vivo Tissue Study Results Tissue Profiling by Histology

Variation in tissue quality and cellular composition were monitored intissue sections either after formalin fixation and paraffin embedding orafter tissue freezing and fixation. Overall quality was evaluated afterhematoxylin-eosin staining (HE).

As shown in FIG. 7, Im02 induced histological rearrangements (C), whichwere not seen with an Ad0 control vector (B), or single gene vectors(data not shown). Im02 induced morphological alterations observed intumor tissues (FIG. 7, panel C) include the number and distribution ofimmune cell infiltrates with respect to tissues treated with eithercontrol Ad0/AdNull (panel B) or single-gene vectors (data not shown).

Furthermore, FIG. 8 shows that Im02 induces apparent histologicalrearrangements in the distribution and frequency of leukocyteinfiltrates. The arrow in the lower panel of FIG. 8 indicates a tumorregion with signs of cell death. These morphological changes providehistological evidence for an Im02-induced immune response.

Identification of Target Cell Types

Transmission electron microscopy (TEM) on tissues transduced with Im02was performed to identify, inter alia, the target cell type ofadenoviral particle uptake, the route of uptake, judged by the presenceand morphology of vesicle membranes, and the abundance of particles percell.

Tumor tissue from a cystectomy was dissected and transduced bysubmerging the sample with 500 μl culture medium containing 10⁸ ivp Im02for 1 hour at 37° C., uptake was stopped by medium replacement withice-cold fixation solution containing 2% glutaraldehyde. Tissue sampleswere then processed by standard procedures and images taken bytransmission electron microscopy, supplied by Vironova SA, Stockholm,Sweden.

In positive stain transmission electron microscopy, adenoviral particleswere identified by their size of about 80 nm and by particle geometry.The results are shown in FIG. 9. After one hour of transduction, Im02adenoviral particles are detectable in a variety of cell types asidentified by ultrastructural morphology analyses. Presence andmorphology of vesicle structures around adenoviral particles indicatethe uptake mechanism.

By their individual morphology, different cell types were found astarget cells in bladder carcinoma samples, including tumor cells (notshown), connective tissue cells (fibroblast, Panel 4) of the tumorstroma and immune cells. Adenoviral particles were detected in cells oflymphocyte (Panel 3) and monocyte morphology (Panel 2). In bladdercarcinoma, monocyte morphology indicates presence of Langerhans cells,macrophages or dendritic cells.

Importantly, this finding is of particular value for the mode-of-action,since all these identified target immune cells are described to undergoactivation and differentiation into effector cell types aftertransduction with cytokines, while 4-1BBL-expression was shown tosupport activating processes when expressed in antigen-presenting cellsand on lymphocytes, by reverse signal transduction (Ju et al., 2009,International Immunology, 21(10), 1135-1144). Route of uptake andabundance: Adenoviral particles were found in the cytoplasm of targetcells in different positions and with different surroundings. Theclassical uptake is mediated after binding to the Coxsackie andAdenovirus receptor, followed by shuttling in endocytotic vesicles.

This pathway is suggested to be active in Panel 6A, where a particle isimaged in the process of vesicle formation. In Panel 6B, an adenoviralparticle is located in a large vesicle also containing other non-definedstructures, suggesting a pinocytotic way of uptake. In Panel 3A, anadenoviral particle was captured in a circular membrane structuresuggesting advanced endocytotic uptake.

Exposure of tissue samples to Im02 for only one hour was chosen inaccordance with a future intravesicular instillation protocol. In ourtissue model particles reach areas at a depth of several cell layers.Regularly groups of up to 30 adenoviral particles per cell wereidentified (Panel 5A).

Example 9: Comparison of Differential Expression in Normal Bladder andBladder Tumor Samples

Normal bladder and tumor tissues were transduced with 10⁸ ivp Im02.Cultures were continued until day 6. Differential expression wasdetermined in comparison to samples treated with Ad0 (empty adenoviralvector) as a control. FIG. 10 shows the results for a pool of 9 bladderand 10 tumor tissue samples. The overall stimulation (i.e., induction ofimmune response processes) is higher in tumor tissues than in normalbladder tissues (see FIG. 10). This effect points on differences in themicroenvironment between normal and tumor tissues, e.g., differences inimmune cell infiltrate numbers or the level of suppression in thevicinity to tumor cells.

Example 10: Examination of Transfectants for Adenoviral Uptake

Adenoviral uptake into intact bladder tissue can be improved by theaddition of transfectant-like polycationic compounds. For ex vivo tissuesample perfusion of Im02, protamine sulfate (10 μg/ml) was identified toenable adenoviral product uptake independent ofCoxsackie-Adenovirus-receptor (CAR) expression in a candidate compoundscreen in a set of cell lines (see FIG. 11). The human bladder carcinomaline RT-4 reportedly expresses CAR, whereas the mouse colon carcinomaline CT-26 does not. The adenoviral product was formulated in thebuffers as indicated in the legend of FIG. 11. Human bladder cancer cellline RT-4 and the CAR-negative murine colon cancer cell line CT-26 weretransduced with an Ad-GFP (adenoviral vector carrying GFP) at differentmultiplicities per target cell (MOI: multiplicity of infection). Resultsare illustrated as percentage of GFP-positive cells 48 hours aftertransduction, measured by flow cytometry. As a result, in both celllines protamine sulfate at 10 μg/ml enabled highest transductionefficiency.

Abbreviations: Merck Buffer: 5 mM Tris pH 8.0, 75 mM NaCl, 5% Sucrose,0.005% Polysorbate 80, 1 mM MgCl₂; all further additions are formulatedin Merck Buffer; Chitosan: 1%, Mannitol: 1 M; Protamine: 10 μg/mlprotamine sulfate, Pluronic F68: 0.001%; Blended: a blend of Sucrose,Mannitol and Pluronic F68. Merck-buffer indicates the basic formulationwithout supporting transfectant additives. Presence of CAR is indicatedby sufficient transduction at low multiplicity of infection (MOI,infectious viral particles per target cell). In the absence of CAR,uptake is achieved only by low affinity uptake via integrin-mediateduptake. The effect without additive is illustrated in RT-4 (approx. 25%transduction at MOI 100) versus CT-26 (<5% transduction at MOI 5000).Addition of 10 μg/ml protamine sulfate doubles transduction in RT-4 andtriples it in CT-26.

A cooperative adjuvant effect of protamine sulfate was also found in theex vivo tissue culture (see FIG. 12). Tumor and bladder tissues weretransduced with 10⁸ ivp Im02 or Ad0 (empty adenoviral vector) with orwithout addition of 10 μg/ml protamine sulfate. Transgene expression andIFN-γ expression as response cytokine was measured at day 6 aftertransduction in the culture supernatant by ELISA. In the presence of 10μg/ml protamine sulfate, the expression of the response cytokine IFN-γis higher at even lower transgene expression, which suggests that Im02in the presence of protamine sulfate stimulates more immune cells.

Based on results on the panel of cell lines and the evaluation of targetcell condition, this formulation was applied in the ex vivo therapysimulation study.

Example 11: Transgene Expression of 4-1BBL, IL-2, and scIL-12 of Im02and of Single-Gene Expressing Vectors at Different MOI, and IFN-γResponse

Transgene expression of 4-1BBL, IL-2 and scIL-12 and IFN-γ response ofIm02, and combinations of single-gene expressing vectors reveal thatIFN-γ expression is dependent on increasing 4-1BBL levels.

Human A549-cells were transduced for one hour with Im02 or combinationsof single-gene adenoviral vectors expressing scIL-12, IL-2, and 4-1BBL,respectively, at multiplicities of infection (MOI, i.e., infectiousviral particles per target cell) as indicated by numbers in brackets(see FIG. 19). Human peripheral blood mononuclear cells (PBMCs) wereadded 4 hours after transduction and supernatants were collected forcytokine assays after 34 hours of co-culture. Cytokine levels weredetected by ELISA. Tumor cells were detached and cells positive for4-1BBL were detected by flow cytometry. Indicated data points are themean of four individual donors, each with four replicates.

As shown in FIG. 19, single-gene vectors expressing IL-12, IL-2, and4-1BBL alone at MOI [5] lead to induction of basic levels of up to 4.2ng/ml IFN-γ. A combination of IL-12 and IL-2 does not significantlyincrease this level. Combinations of constant levels of IL-12 and IL-2,both at MOI [5], with increasing levels of 4-1BBL up to MOI [100], leadto increasing IFN-γ induction at moderate IL-12 levels.

It is important to note that the IL-12 expression must not be increasedunlimitedly as expressed by the vector Im01 described in Example 3 (seeFIG. 3). High IL-12 expression is considered to induce down-regulationof immune activation and to cause toxicity. This condition of high IFN-γand moderate IL-12 expression is fulfilled by the vector of the presentdisclosure, in particular by the arrangement of 4-1BBL, IL-2, and IL-12shown in vector Im02.

1. A method of treating cancer, a viral infection and/or an immunesystem disorder, comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a vector, wherein the vector comprises (a) nucleic acidsequences encoding 4-1BB ligand (4-1BBL), single chain IL-12 (scIL-12)and IL-2, and (b) at least one regulatory nucleic acid sequenceproviding for an increased expression level of 4-1BBL as compared to theexpression levels of scIL-12 and IL-2.
 2. The method of claim 1, whereinthe expression level of 4-1BBL is increased as compared to theexpression level of 4-1BBL obtained by the expression construct ofvector Im01.
 3. The method of claim 1, wherein the expression level ofscIL-12 is decreased and/or the expression level of IL-2 is increased ascompared to the expression levels of scIL-12 and/or IL-2 obtained by theexpression construct of vector Im01.
 4. The method of claim 1, whereinthe nucleic acid sequences encoding 4-1BBL, scIL-12 and IL-2 areorganized in 5′ to 3′ orientation in a sequential order 1, 2, 3, withthe proviso that the sequence encoding scIL-12 is not at position
 1. 5.The method of claim 1, wherein the vector is any one of an adenoviralvector, an adeno-associated virus vector, a lentiviral vector, a herpessimplex virus vector, a pox virus vector, an RNA vector, a plasmidvector, a nanoparticle vector, and naked DNA.
 6. The method of claim 1,wherein the nucleic acid sequence encoding 4-1BBL is human cDNA, thenucleic acid sequence encoding scIL-12 is human cDNA, and/or the nucleicacid sequence encoding IL-2 is human cDNA.
 7. The method of claim 1,wherein the nucleic acid sequence encoding 4-1BBL shows at least 70%sequence identity to the nucleic acid sequence of SEQ ID NO: 1, whereinthe variant nucleic acid sequence encodes a 4-1BBL protein capable ofspecifically binding activated T cells.
 8. The method of claim 1,wherein the nucleic acid sequence encoding IL-2 shows at least 70%sequence identity to the nucleic acid sequence of SEQ ID NO: 3, whereinthe variant nucleic acid sequence encodes an IL-2 protein having immunestimulating activity.
 9. The method of claim 1, wherein the nucleic acidsequence encoding scIL-12 shows at least 70% sequence identity to thenucleic acid sequence of SEQ ID NO: 5, wherein the variant nucleic acidsequence encodes a scIL-12 protein having immune stimulating activity.10. The method of claim 1, wherein the nucleic acid sequences encodingscIL-12 and IL-2 are located downstream of the nucleic acid sequenceencoding 4-1BBL.
 11. The method of claim 10, wherein the nucleic acidsequence encoding IL-2 is located downstream of the nucleic acidsequence encoding 4-1BBL, and the nucleic acid sequence encoding scIL-12is located downstream of the nucleic acid sequence encoding IL-2. 12.The method of claim 10, wherein a promoter is located upstream of thenucleic acid sequence encoding 4-1BBL, but not upstream of the nucleicacid sequences encoding scIL-12 and/or IL-2.
 13. The method of claim 1,wherein the nucleic acid sequences encoding 4-1BBL, scIL-12 and IL-2 arelinked by internal ribosomal entry sites (IRES).
 14. The method of claim1, wherein the at least one regulatory nucleic acid sequence is at leastone promoter sequence.
 15. The method of claim 1, wherein the cancer isany one of bladder cancer, breast cancer, prostate cancer, lymphoma,skin cancer, pancreatic cancer, colon cancer, melanoma, malignantmelanoma, ovarian cancer, brain cancer, primary brain carcinoma,head-neck cancer, glioma, glioblastoma, liver cancer, non-small celllung cancer, head or neck carcinoma, breast carcinoma, ovariancarcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor,cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreaticcarcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma,genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma,myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma,endometrial carcinoma, adrenal cortex carcinoma, malignant pancreaticinsulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosisfungoides, malignant hypercalcemia, cervical hyperplasia, leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic granulocyticleukemia, acute granulocytic leukemia, hairy cell leukemia,neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera,essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,soft-tissue sarcoma, mesothelioma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.
 16. The method of claim 1, wherein themethod is a method of treating cancer metastasis.